http://2012.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=250&target=M.B.ZHOU2012.igem.org - User contributions [en]2024-03-28T08:37:10ZFrom 2012.igem.orgMediaWiki 1.16.0http://2012.igem.org/Team:SUSTC-Shenzhen-A/Biodesign_TutorialTeam:SUSTC-Shenzhen-A/Biodesign Tutorial2012-10-27T03:57:32Z<p>M.B.ZHOU: </p>
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<td><div><br />
<br />
<h1 class="title">&nbsp;BioDesign Tutorial</h1><br />
<br />
<br />
<a name="Video" ></a> <br />
<h3 class="title1">&nbsp;&nbsp;Video tutorial</h3><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/Devidingline_whole.jpg"/><br />
<br />
<p align="center"><br />
<embed src="http://player.youku.com/player.php/sid/XNDY3Mzg4Njcy/v.swf" <br />
width="480" height="400" <br />
type="application/x-shockwave-flash"><br />
</embed></p><br />
<p align="center">&nbsp;&nbsp;Note: There will be a 6-second Chinese ad at the beginning of this video.</p><br />
<br />
<h3 class="title1">&nbsp;&nbsp;Step by step instruction</h3><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/Devidingline_whole.jpg"/><br />
<ul><br />
<a href="#Folder">Folder page <br />
</a> &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
<br />
<a href="#File">File page <br />
</a>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
<br />
<a href="#Drawing">Drawing page <br />
</a>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
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<a href="#Mail">Mail page <br />
</a> &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
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<td>&nbsp;</td><br />
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<tr><td><a name="Folder" ></a> <br />
<p class="title1">&nbsp;&nbsp;Folder page</p><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/Devidingline_whole.jpg"/><br />
<br />
<p><img src="https://static.igem.org/mediawiki/2012/e/e6/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_1.png" valign="top" align="left" width="300" height = "480" style="BORDER:#CCFFCC 5px dashed;margin:10px;" ><img src="https://static.igem.org/mediawiki/2012/b/b7/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_2.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:10px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(1)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Fig.1 is the first page. </p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;In order to create a new project/folder, you should click the ‘add’ button at the top right (Fig.2).</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(2)--></p><br />
<p>&nbsp;&nbsp;</p><br />
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<p><img src="https://static.igem.org/mediawiki/2012/3/35/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_3.png" valign="top" align="left" height="480" width="300" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(3)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the icon, then click ‘enter’,<br/> it goes into the secondary page (Fig.3).</p><br />
<p>&nbsp;&nbsp;</p><br />
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<td><a name="File" ></a> <br />
<p class="title1">&nbsp;&nbsp;File page</p><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/Devidingline_whole.jpg"/><br />
<p><img src="https://static.igem.org/mediawiki/2012/2/26/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_4.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/6/60/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_5.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:10px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(4)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;The secondary page looks like Fig.4. </p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the button name ‘add’ at the top right to create a new file (Fig.5).</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(5)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
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<a name="Drawing"></a><br />
<p class="title1">&nbsp;&nbsp;Drawing page</p><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/Devidingline_whole.jpg"/><br />
<p><img src="https://static.igem.org/mediawiki/2012/4/41/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_6.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/5/54/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_7.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(6)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the newly created file, choose ‘edit’ to do the drawing (Fig.6).</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click a object at the bottom to set it on the screen.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(7)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
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<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/8/8f/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_8.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/b/b2/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_9.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(8)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the text field next to a part to rename this part.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;For the objects in “comp”, you can zoom them by ywo fingers to change the their sizes.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(9)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
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<br/><br />
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</td><br />
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<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/1/14/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_10.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/f/f6/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_11.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(10)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can rotate the object clockwisely by clicking the rotating button.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can get a vector on the canvas from “part”.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(11)--></p><br />
<p>&nbsp;&nbsp;</p><br />
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<p><img src="https://static.igem.org/mediawiki/2012/f/fd/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_12.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/c/c8/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_13.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(12)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can change the size of the vector by zooming with two fingers.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;If you have add a cell, you may find it covers the vector or other object. Click the “down” button to lower layer.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(13)--></p><br />
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<p><img src="https://static.igem.org/mediawiki/2012/6/63/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_14.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/7/71/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_15.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(14)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can put objects(in”mole. & part.”) on vector by draging it into the vector.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
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<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;In “reac.”, you have various choices to connect objects by Bezier curve.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(15)--></p><br />
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<p><img src="https://static.igem.org/mediawiki/2012/2/24/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_16.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/f/fa/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_17.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(16)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click two objects in turn and they will be connected.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can click the place near the line to pitch the curve. And then change the shape of the curve by dragging with one or two fingers.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(17)--></p><br />
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<p><img src="https://static.igem.org/mediawiki/2012/c/cb/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_18.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/9/99/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_19.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(18)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can change the type of the arrow by clicking the first button at the right.</p><br />
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<p>&nbsp;&nbsp;Fig.19 shows the changed arrow.</p><br />
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<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(19)--></p><br />
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<p><img src="https://static.igem.org/mediawiki/2012/7/77/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_20.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/0/0e/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_21.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(20)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can pitch the center point to edit both parts at the same time.</p><br />
<p>&nbsp;&nbsp;</p><br />
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<p>&nbsp;&nbsp;In “regu.”, choose one to connect two or three parts by polygonal line.</p><br />
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<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(21)--></p><br />
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<p><img src="https://static.igem.org/mediawiki/2012/c/ca/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_22.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/0/00/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_23.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(22)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Fig.22 shows what happens after choosing the "regu".</p><br />
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<p>&nbsp;&nbsp;Fig.23 also shows what happens after choosing the "regu".</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(23)--></p><br />
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<p><img src="https://static.igem.org/mediawiki/2012/6/66/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_24.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/6/6d/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_25.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(24)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;For objects in “part.”, you can move one group close to another to connet them.</p><br />
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<p>&nbsp;&nbsp;You can press the button to disconnect them.</p><br />
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<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(25)--></p><br />
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<p><img src="https://static.igem.org/mediawiki/2012/d/df/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_70.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/9/90/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_71.png" valign="top" align="right" width="300"height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(26)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the button at the top right to save this picture (Fig.26). </p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Come back, you’ll see the icon has change into the saved picture (Fig.27).</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(27)--></p><br />
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<p><img src="https://static.igem.org/mediawiki/2012/e/ef/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_72.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/f/f8/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_73.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(28)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the microscope button in FIle page at the bottom to search a file (Fig.28). </p><br />
<p>&nbsp;&nbsp;</p><br />
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<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
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<p>&nbsp;&nbspClick the button at the bottom right to present the files in a table (Fig.29).</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(29)--></p><br />
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<td><a name="Mail" ></a> <br />
<p class="title1">&nbsp;&nbsp;Mail page</p><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/Devidingline_whole.jpg" /><br />
<p><img src="https://static.igem.org/mediawiki/2012/b/bf/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_74.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/5/5a/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_75.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:10px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(30)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Come back to the Folder page. Click the envelope button at the bottom right to the file that you want to email to your friend! (Fig.30)</p><br />
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<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the ‘done’ button. Send the chosen files to your friends (Fig.31)!</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(31)--></p><br />
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[[file:footbar.jpg|center]]</div>M.B.ZHOUhttp://2012.igem.org/Team:SUSTC-Shenzhen-A/User_ReviewTeam:SUSTC-Shenzhen-A/User Review2012-10-27T03:56:35Z<p>M.B.ZHOU: </p>
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<h1 class="title">&nbsp;&nbsp;<strong>User Review</strong></h1><br />
<p>&nbsp;Our software is very popular among iGEM teams and synthetic biologists. There are more than 20 users in less than one week time after we released Beta version of the software in Asia. The user distribution in Asia is shown in the map. We did a survey about comments on Partsregistry and our software. The result is listed below.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title1">&nbsp;&nbsp;Users Around the World</p><br />
<p>&nbsp;&nbsp;Since the time our BioSearch has been launched on APP Store, our clients around the world had used this software!<br />
The figure below shows the user distribution. Green place indicates the country that the user locates.</p><br />
<p>&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2012/c/c3/Sustc_shenzhen_a_Wordmap.png"align="center"></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;In order to satisfy the need of major iGEM participants and synthetic biologists, SUSTC-Shenzhen-A send a survey to several iGEM teams in mainland China to collect their opinions on Partsregistry and our App. You can see from the following figure, teams from <B><I>Ocean University of China, Shanghai Jiaotong University, Tianjin University and Tsinghua University</B></I> are very active in our surveys.They highly think of our project and provide us with a lot of precious suggestions. Here we want to express our appriciation to them from the deep heart.</p><br />
<p>&nbsp;&nbsp;</p><br />
<img src="https://static.igem.org/mediawiki/2012/8/8e/SUSTC_UR1.jpg"width=440><br />
<img src="https://static.igem.org/mediawiki/2012/9/9b/SUSTC_UR2.jpg"width=440><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p><strong>&nbsp;&nbsp;&nbsp;&nbsp;Question1: Registry of standard biological parts(http://partsregistry.org) provides users with a search bar on the top of the right corner. Are you satisfied with its searching result?</strong></p><br />
<p><strong>&nbsp;&nbsp;&nbsp;&nbsp;Question2: Will you expect us to make Partsregistry an iPhone App?</strong></p><br />
<br><br />
<br />
<br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;The figure shows that 8% users feel unsatisfactory about Partsregistry, 46% feel acceptable, 46% feel satisfactory about it.Therefore, it's our responsibility to make it more convenient for researchers to search for their desired biobricks. Another survey shows that over 85% of iGEM participants are in an urgent need for an iPhone App about Partsregistry. That strongly demonstrate the significance to proceed this project. </p><br />
<br><br />
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<img src="https://static.igem.org/mediawiki/2012/6/62/SUSTC_UR3.jpg"width=440><br />
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<p><strong>&nbsp;&nbsp;&nbsp;&nbsp;Question3: How do you evaluate the search function in BioSearch according to your firsthand user experience?<br />
</strong></p><br />
<p><strong>&nbsp;&nbsp;&nbsp;&nbsp;Question4:How do you think of our effort in optimizing page layout and human oriented design?<br />
</strong></p><br />
<br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;The figure shows that more than 60% of the users think our software is both functional and beautiful.</p><br />
<br><br />
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<p>&nbsp;&nbsp;</p><br />
<p class="title1">&nbsp;&nbsp;User Comments</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Rachel J.K </strong></p><br />
<p>&nbsp;&nbsp;Very handy tool! Bought professional work to everyday life...</p><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>alphatenz </strong></p><br />
<p>&nbsp;&nbsp;It's an amazing app!</p><br />
<br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Mickey Donkey </strong></p><br />
<p>&nbsp;&nbsp;I hope this app can make my job more convenient, thank you developers!</p><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Karen890 </strong></p><br />
<p>&nbsp;&nbsp;Biosearch has a clear and concise user interface and its search function is quiet amazing! The results of fuzzy search are really useful and even well <br/>&nbsp;&nbsp;arranged. I can also visit partsregistry from hyperlinks. It will be better if an iPad version is provided.</p><br />
<br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>austinamens </strong>from Tianjin</p><br />
<p>&nbsp;&nbsp;I searched by keywords and by part name. BioSearch is indeed a good trying.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong> jiaquan_good </strong>from Tianjin.</p><br />
<p>&nbsp;&nbsp;I looked over bioparts list by category. It is easy to use and the tutorial is very helpful.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>huanan1991 </strong>from SJTU-BioX-Shanghai</p><br />
<p>&nbsp;&nbsp;I searched and viewed several lists by catalog. I use BioaSearch to search bioparts relative to synthesis of fatty acid.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Adjaniyu</strong> from SJTU-BioX-Shanghai</p><br />
<p>&nbsp;&nbsp;I’m very excited to see such a pithy user interface. I searched several vio-operon parts.<p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Vampire19</strong> from OUC-iGEM</p><br />
<p>&nbsp;&nbsp;I searched BioBrick J23015 and clicked the link in BioSearch to view experience page on website.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Oucigem1</strong> from OUC-China</p><br />
<p>&nbsp;&nbsp;The overall effect of BioSearch is good. I searched BBa_K116401 and BBa_J23101.<p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Newfive </strong>from OUC-China<p><br />
<p>&nbsp;&nbsp;With BioSearch, I searched BBa_K737005 and added it to “Favorite”.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Pengyongouc </strong>from OUC-China</p><br />
<p>&nbsp;&nbsp;I searched Bba_K737067 and got expecting result.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>cwth166</strong> from OUC-China</p><br />
<p>&nbsp;&nbsp;I searched J23106, R0011 and E0480. It is fast and accurate, and it’s very beautiful.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Oucigem2 </strong> from OUC-China</p><br />
<p>&nbsp;&nbsp;As for its search function, BioSearch is strong and accurate, though it is not a multi-function. I searched BBa-J23106. </p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Liuerlrir </strong> from OUC-China</p><br />
<p>&nbsp;&nbsp;I tried all the function of BioSearch and searched BBa_R0082, cph8 and k081024. I thought I got what I wanted.</p><br />
<p>&nbsp;&nbsp;<p><br />
<p>&nbsp;&nbsp;<strong>530854106QQ</strong> from OUC-China</p><br />
<p>&nbsp;&nbsp;I searched Bba_K737067 with BioSearch on iPhone which is much faster than Partsregistry. I really expect you to develop an iPad version.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>kaykang510</strong> from Tsinghua</p><br />
<p>&nbsp;&nbsp;I tried search by name, by keywords, by type and by catalog. I scanned sub list of LuxI, LuxR, Las and FemE.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>astrol.lee</strong> from Tsinghua</p><br />
<p>&nbsp;&nbsp;I viewed the lists of RFP, cI and lacI. It’s fast and accurate.</p><br />
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[[file:footbar.jpg|center]]</div>M.B.ZHOUhttp://2012.igem.org/Team:SUSTC-Shenzhen-A/User_ReviewTeam:SUSTC-Shenzhen-A/User Review2012-10-27T03:54:00Z<p>M.B.ZHOU: </p>
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<table width="899" border="0" cellspacing="0px" cellpadding="0px"align="center"><br />
<tr><br />
<td valign="top"><br />
<h1 class="title">&nbsp;&nbsp;<strong>User Review</strong></h1><br />
<p>&nbsp;Our software is very popular among iGEM teams and synthetic biologists. There are more than 20 users in less than one week time after we released Beta version of the software in Asia. The user distribution in Asia is shown in the map. We did a survey about comments on Partsregistry and our software. The result is listed below.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title1">&nbsp;&nbsp;Users Around the World</p><br />
<p>&nbsp;&nbsp;Since the time our BioSearch has been launched on APP Store, our clients around the world had used this software!<br/><br />
The figure below shows the user distribution. Green place indicates the country that the user locates.</p><br />
<p>&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2012/c/c3/Sustc_shenzhen_a_Wordmap.png"align="center"></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;In order to satisfy the need of major iGEM participants and synthetic biologists, SUSTC-Shenzhen-A send a survey to several iGEM teams in mainland China to collect their opinions on Partsregistry and our App. You can see from the following figure, teams from <B><I>Ocean University of China, Shanghai Jiaotong University, Tianjin University and Tsinghua University</B></I> are very active in our surveys.They highly think of our project and provide us with a lot of precious suggestions. Here we want to express our appriciation to them from the deep heart.</p><br />
<p>&nbsp;&nbsp;</p><br />
<img src="https://static.igem.org/mediawiki/2012/8/8e/SUSTC_UR1.jpg"width=440><br />
<img src="https://static.igem.org/mediawiki/2012/9/9b/SUSTC_UR2.jpg"width=440><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p><strong>&nbsp;&nbsp;&nbsp;&nbsp;Question1: Registry of standard biological parts(http://partsregistry.org) provides users with a search bar on the top of the right corner. Are you satisfied with its searching result?</strong></p><br />
<p><strong>&nbsp;&nbsp;&nbsp;&nbsp;Question2: Will you expect us to make Partsregistry an iPhone App?</strong></p><br />
<br><br />
<br />
<br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;The figure shows that 8% users feel unsatisfactory about Partsregistry, 46% feel acceptable, 46% feel satisfactory about it.Therefore, it's our responsibility to make it more convenient for researchers to search for their desired biobricks. Another survey shows that over 85% of iGEM participants are in an urgent need for an iPhone App about Partsregistry. That strongly demonstrate the significance to proceed this project. </p><br />
<br><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/6/62/SUSTC_UR3.jpg"width=440><br />
<img src="https://static.igem.org/mediawiki/2012/5/5c/SUSTC_UR4.jpg"width=440><br />
<br />
<p><strong>&nbsp;&nbsp;&nbsp;&nbsp;Question3: How do you evaluate the search function in BioSearch according to your firsthand user experience?<br />
</strong></p><br />
<p><strong>&nbsp;&nbsp;&nbsp;&nbsp;Question4:How do you think of our effort in optimizing page layout and human oriented design?<br />
</strong></p><br />
<br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;The figure shows that more than 60% of the users think our software is both functional and beautiful.</p><br />
<br><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title1">&nbsp;&nbsp;User Comments</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Rachel J.K </strong></p><br />
<p>&nbsp;&nbsp;Very handy tool! Bought professional work to everyday life...</p><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>alphatenz </strong></p><br />
<p>&nbsp;&nbsp;It's an amazing app!</p><br />
<br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Mickey Donkey </strong></p><br />
<p>&nbsp;&nbsp;I hope this app can make my job more convenient, thank you developers!</p><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Karen890 </strong></p><br />
<p>&nbsp;&nbsp;Biosearch has a clear and concise user interface and its search function is quiet amazing! The results of fuzzy search are really useful and even well <br/>&nbsp;&nbsp;arranged. I can also visit partsregistry from hyperlinks. It will be better if an iPad version is provided.</p><br />
<br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>austinamens </strong>from Tianjin</p><br />
<p>&nbsp;&nbsp;I searched by keywords and by part name. BioSearch is indeed a good trying.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong> jiaquan_good </strong>from Tianjin.</p><br />
<p>&nbsp;&nbsp;I looked over bioparts list by category. It is easy to use and the tutorial is very helpful.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>huanan1991 </strong>from SJTU-BioX-Shanghai</p><br />
<p>&nbsp;&nbsp;I searched and viewed several lists by catalog. I use BioaSearch to search bioparts relative to synthesis of fatty acid.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Adjaniyu</strong> from SJTU-BioX-Shanghai</p><br />
<p>&nbsp;&nbsp;I’m very excited to see such a pithy user interface. I searched several vio-operon parts.<p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Vampire19</strong> from OUC-iGEM</p><br />
<p>&nbsp;&nbsp;I searched BioBrick J23015 and clicked the link in BioSearch to view experience page on website.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Oucigem1</strong> from OUC-China</p><br />
<p>&nbsp;&nbsp;The overall effect of BioSearch is good. I searched BBa_K116401 and BBa_J23101.<p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Newfive </strong>from OUC-China<p><br />
<p>&nbsp;&nbsp;With BioSearch, I searched BBa_K737005 and added it to “Favorite”.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Pengyongouc </strong>from OUC-China</p><br />
<p>&nbsp;&nbsp;I searched Bba_K737067 and got expecting result.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>cwth166</strong> from OUC-China</p><br />
<p>&nbsp;&nbsp;I searched J23106, R0011 and E0480. It is fast and accurate, and it’s very beautiful.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Oucigem2 </strong> from OUC-China</p><br />
<p>&nbsp;&nbsp;As for its search function, BioSearch is strong and accurate, though it is not a multi-function. I searched BBa-J23106. </p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Liuerlrir </strong> from OUC-China</p><br />
<p>&nbsp;&nbsp;I tried all the function of BioSearch and searched BBa_R0082, cph8 and k081024. I thought I got what I wanted.</p><br />
<p>&nbsp;&nbsp;<p><br />
<p>&nbsp;&nbsp;<strong>530854106QQ</strong> from OUC-China</p><br />
<p>&nbsp;&nbsp;I searched Bba_K737067 with BioSearch on iPhone which is much faster than Partsregistry. I really expect you to develop an iPad version.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>kaykang510</strong> from Tsinghua</p><br />
<p>&nbsp;&nbsp;I tried search by name, by keywords, by type and by catalog. I scanned sub list of LuxI, LuxR, Las and FemE.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>astrol.lee</strong> from Tsinghua</p><br />
<p>&nbsp;&nbsp;I viewed the lists of RFP, cI and lacI. It’s fast and accurate.</p><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br />
</td><br />
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[[file:footbar.jpg|center]]</div>M.B.ZHOUhttp://2012.igem.org/Team:SUSTC-Shenzhen-A/User_ReviewTeam:SUSTC-Shenzhen-A/User Review2012-10-27T03:52:34Z<p>M.B.ZHOU: </p>
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<body ><br />
<br />
<table width="899" border="0" cellspacing="0px" cellpadding="0px"align="center"><br />
<tr><br />
<td valign="top"><br />
<h1 class="title">&nbsp;&nbsp;<strong>User Review</strong></h1><br />
<p>&nbsp;Our software is very popular among iGEM teams and synthetic biologists. There are more than 20 users in less than one week time after we released Beta version of the software in Asia. The user distribution in Asia is shown in the map. We did a survey about comments on Partsregistry and our software. The result is listed below.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title1">&nbsp;&nbsp;Users Around the World</p><br />
<p>&nbsp;&nbsp;Since the time our BioSearch has been launched on APP Store, our clients around the world had used this software!<br/><br />
The figure below shows the user distribution. Green place indicates the country that the user locates.</p><br />
<p>&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2012/c/c3/Sustc_shenzhen_a_Wordmap.png"align="center"></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;In order to satisfy the need of major iGEM participants and synthetic biologists, SUSTC-Shenzhen-A send a survey to several iGEM teams in mainland China to collect their opinions on Partsregistry and our App. You can see from the following figure, teams from <B><I>Ocean University of China, Shanghai Jiaotong University, Tianjin University and Tsinghua University</B></I> are very active in our surveys.They highly think of our project and provide us with a lot of precious suggestions. Here we want to express our appriciation to them from the deep heart.</p><br />
<p>&nbsp;&nbsp;</p><br />
<img src="https://static.igem.org/mediawiki/2012/8/8e/SUSTC_UR1.jpg"width=440><br />
<img src="https://static.igem.org/mediawiki/2012/9/9b/SUSTC_UR2.jpg"width=440><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p><strong>&nbsp;&nbsp;&nbsp;&nbsp;Question1: Registry of standard biological parts(http://partsregistry.org) provides users with a search bar on the top of the right corner. Are you satisfied with its searching result?</strong></p><br />
<p><strong>&nbsp;&nbsp;&nbsp;&nbsp;Question2: Will you expect us to make Partsregistry an iPhone App?</strong></p><br />
<br><br />
<br />
<br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;The figure shows that 8% users feel unsatisfactory about Partsregistry, 46% feel acceptable, 46% feel satisfactory about it.Therefore, it's our responsibility to make it more convenient for researchers to search for their desired biobricks. Another survey shows that over 85% of iGEM participants are in an urgent need for an iPhone App about Partsregistry. That strongly demonstrate the significance to proceed this project. </p><br />
<br><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/6/62/SUSTC_UR3.jpg"width=440><br />
<img src="https://static.igem.org/mediawiki/2012/5/5c/SUSTC_UR4.jpg"width=440><br />
<br />
<p><strong>&nbsp;&nbsp;&nbsp;&nbsp;Question3: How do you evaluate the search function in BioSearch according to your firsthand user experience?<br />
</strong></p><br />
<p><strong>&nbsp;&nbsp;&nbsp;&nbsp;Question4:How do you think of our effort in optimizing page layout and human oriented design?<br />
</strong></p><br />
<br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;The figure shows that more than 60% of the users think our software is both functional and beautiful.</p><br />
<br><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title1">&nbsp;&nbsp;User Comments</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Rachel J.K </strong></p><br />
<p>&nbsp;&nbsp;Very handy tool! Bought professional work to everyday life...</p><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>alphatenz </strong></p><br />
<p>&nbsp;&nbsp;It's an amazing app!</p><br />
<br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Mickey Donkey </strong></p><br />
<p>&nbsp;&nbsp;I hope this app can make my job more convenient, thank you developers!</p><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Karen890 </strong></p><br />
<p>&nbsp;&nbsp;Biosearch has a clear and concise user interface and its search function is quiet amazing! The results of fuzzy search are really useful and even well arranged. I can also visit partsregistry from hyperlinks. It will be better if a iPad version is provided.</p><br />
<br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>austinamens </strong>from Tianjin</p><br />
<p>&nbsp;&nbsp;I searched by keywords and by part name. BioSearch is indeed a good trying.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong> jiaquan_good </strong>from Tianjin.</p><br />
<p>&nbsp;&nbsp;I looked over bioparts list by category. It is easy to use and the tutorial is very helpful.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>huanan1991 </strong>from SJTU-BioX-Shanghai</p><br />
<p>&nbsp;&nbsp;I searched and viewed several lists by catalog. I use BioaSearch to search bioparts relative to synthesis of fatty acid.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Adjaniyu</strong> from SJTU-BioX-Shanghai</p><br />
<p>&nbsp;&nbsp;I’m very excited to see such a pithy user interface. I searched several vio-operon parts.<p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Vampire19</strong> from OUC-iGEM</p><br />
<p>&nbsp;&nbsp;I searched BioBrick J23015 and clicked the link in BioSearch to view experience page on website.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Oucigem1</strong> from OUC-China</p><br />
<p>&nbsp;&nbsp;The overall effect of BioSearch is good. I searched BBa_K116401 and BBa_J23101.<p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Newfive </strong>from OUC-China<p><br />
<p>&nbsp;&nbsp;With BioSearch, I searched BBa_K737005 and added it to “Favorite”.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Pengyongouc </strong>from OUC-China</p><br />
<p>&nbsp;&nbsp;I searched Bba_K737067 and got expecting result.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>cwth166</strong> from OUC-China</p><br />
<p>&nbsp;&nbsp;I searched J23106, R0011 and E0480. It is fast and accurate, and it’s very beautiful.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Oucigem2 </strong> from OUC-China</p><br />
<p>&nbsp;&nbsp;As for its search function, BioSearch is strong and accurate, though it is not a multi-function. I searched BBa-J23106. </p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Liuerlrir </strong> from OUC-China</p><br />
<p>&nbsp;&nbsp;I tried all the function of BioSearch and searched BBa_R0082, cph8 and k081024. I thought I got what I wanted.</p><br />
<p>&nbsp;&nbsp;<p><br />
<p>&nbsp;&nbsp;<strong>530854106QQ</strong> from OUC-China</p><br />
<p>&nbsp;&nbsp;I searched Bba_K737067 with BioSearch on iPhone which is much faster than Partsregistry. I really expect you to develop an iPad version.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>kaykang510</strong> from Tsinghua</p><br />
<p>&nbsp;&nbsp;I tried search by name, by keywords, by type and by catalog. I scanned sub list of LuxI, LuxR, Las and FemE.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>astrol.lee</strong> from Tsinghua</p><br />
<p>&nbsp;&nbsp;I viewed the lists of RFP, cI and lacI. It’s fast and accurate.</p><br />
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[[file:footbar.jpg|center]]</div>M.B.ZHOUhttp://2012.igem.org/Team:SUSTC-Shenzhen-A/User_ReviewTeam:SUSTC-Shenzhen-A/User Review2012-10-27T03:51:19Z<p>M.B.ZHOU: </p>
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<h1 class="title">&nbsp;&nbsp;<strong>User Review</strong></h1><br />
<p>&nbsp;Our software is very popular among iGEM teams and synthetic biologists. There are more than 20 users in less than one week time after we released Beta version of the software in Asia. The user distribution in Asia is shown in the map. We did a survey about comments on Partsregistry and our software. The result is listed below.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title1">&nbsp;&nbsp;Users Around the World</p><br />
<p>&nbsp;&nbsp;Since the time our BioSearch has been launched on APP Store, our clients around the world had used this software!<br/><br />
The figure below shows the user distribution. Green place indicates the country that the user locates.</p><br />
<p>&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2012/c/c3/Sustc_shenzhen_a_Wordmap.png"align="center"></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;In order to satisfy the need of major iGEM participants and synthetic biologists, SUSTC-Shenzhen-A send a survey to several iGEM teams in mainland China to collect their opinions on Partsregistry and our App. You can see from the following figure, teams from <B><I>Ocean University of China, Shanghai Jiaotong University, Tianjin University and Tsinghua University</B></I> are very active in our surveys.They highly think of our project and provide us with a lot of precious suggestions. Here we want to express our appriciation to them from the deep heart.</p><br />
<p>&nbsp;&nbsp;</p><br />
<img src="https://static.igem.org/mediawiki/2012/8/8e/SUSTC_UR1.jpg"width=440><br />
<img src="https://static.igem.org/mediawiki/2012/9/9b/SUSTC_UR2.jpg"width=440><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p><strong>&nbsp;&nbsp;&nbsp;&nbsp;Question1: Registry of standard biological parts(http://partsregistry.org) provides users with a search bar on the top of the right corner. Are you satisfied with its searching result?</strong></p><br />
<p><strong>&nbsp;&nbsp;&nbsp;&nbsp;Question2: Will you expect us to make Partsregistry an iPhone App?</strong></p><br />
<br><br />
<br />
<br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;The figure shows that 8% users feel unsatisfactory about Partsregistry, 46% feel acceptable, 46% feel satisfactory about it.Therefore, it's our responsibility to make it more convenient for researchers to search for their desired biobricks. Another survey shows that over 85% of iGEM participants are in an urgent need for an iPhone App about Partsregistry. That strongly demonstrate the significance to proceed this project. </p><br />
<br><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/6/62/SUSTC_UR3.jpg"width=440><br />
<img src="https://static.igem.org/mediawiki/2012/5/5c/SUSTC_UR4.jpg"width=440><br />
<br />
<p><strong>&nbsp;&nbsp;&nbsp;&nbsp;Question3: How do you evaluate the search function in BioSearch according to your firsthand user experience?<br />
</strong></p><br />
<p><strong>&nbsp;&nbsp;&nbsp;&nbsp;Question4:How do you think of our effort in optimizing page layout and human oriented design?<br />
</strong></p><br />
<br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;The figure shows that more than 60% of the users think our software is both functional and beautiful.</p><br />
<br><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title1">&nbsp;&nbsp;User Comments</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Rachel J.K </strong></p><br />
<p>&nbsp;&nbsp;Very handy tool! Bought professional work to everyday life...</p><br />
<p>&nbsp;&nbsp;</p><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>alphatenz </strong></p><br />
<p>&nbsp;&nbsp;It's an amazing app!</p><br />
<p>&nbsp;&nbsp;</p><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Mickey Donkey </strong></p><br />
<p>&nbsp;&nbsp;I hope this app can make my job more convenient, thank you developers!</p><br />
<p>&nbsp;&nbsp;</p><br />
<br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>austinamens </strong>from Tianjin</p><br />
<p>&nbsp;&nbsp;I searched by keywords and by part name. BioSearch is indeed a good trying.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong> jiaquan_good </strong>from Tianjin.</p><br />
<p>&nbsp;&nbsp;I looked over bioparts list by category. It is easy to use and the tutorial is very helpful.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>huanan1991 </strong>from SJTU-BioX-Shanghai</p><br />
<p>&nbsp;&nbsp;I searched and viewed several lists by catalog. I use BioaSearch to search bioparts relative to synthesis of fatty acid.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Adjaniyu</strong> from SJTU-BioX-Shanghai</p><br />
<p>&nbsp;&nbsp;I’m very excited to see such a pithy user interface. I searched several vio-operon parts.<p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Vampire19</strong> from OUC-iGEM</p><br />
<p>&nbsp;&nbsp;I searched BioBrick J23015 and clicked the link in BioSearch to view experience page on website.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Oucigem1</strong> from OUC-China</p><br />
<p>&nbsp;&nbsp;The overall effect of BioSearch is good. I searched BBa_K116401 and BBa_J23101.<p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Newfive </strong>from OUC-China<p><br />
<p>&nbsp;&nbsp;With BioSearch, I searched BBa_K737005 and added it to “Favorite”.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Pengyongouc </strong>from OUC-China</p><br />
<p>&nbsp;&nbsp;I searched Bba_K737067 and got expecting result.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>cwth166</strong> from OUC-China</p><br />
<p>&nbsp;&nbsp;I searched J23106, R0011 and E0480. It is fast and accurate, and it’s very beautiful.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Oucigem2 </strong> from OUC-China</p><br />
<p>&nbsp;&nbsp;As for its search function, BioSearch is strong and accurate, though it is not a multi-function. I searched BBa-J23106. </p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Liuerlrir </strong> from OUC-China</p><br />
<p>&nbsp;&nbsp;I tried all the function of BioSearch and searched BBa_R0082, cph8 and k081024. I thought I got what I wanted.</p><br />
<p>&nbsp;&nbsp;<p><br />
<p>&nbsp;&nbsp;<strong>530854106QQ</strong> from OUC-China</p><br />
<p>&nbsp;&nbsp;I searched Bba_K737067 with BioSearch on iPhone which is much faster than Partsregistry. I really expect you to develop an iPad version.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>kaykang510</strong> from Tsinghua</p><br />
<p>&nbsp;&nbsp;I tried search by name, by keywords, by type and by catalog. I scanned sub list of LuxI, LuxR, Las and FemE.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>astrol.lee</strong> from Tsinghua</p><br />
<p>&nbsp;&nbsp;I viewed the lists of RFP, cI and lacI. It’s fast and accurate.</p><br />
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[[file:footbar.jpg|center]]</div>M.B.ZHOUhttp://2012.igem.org/Team:SUSTC-Shenzhen-A/Medal_fulfillmentTeam:SUSTC-Shenzhen-A/Medal fulfillment2012-10-27T03:47:09Z<p>M.B.ZHOU: </p>
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<h1 class="title"><strong>Medal Fulfillment</strong></h1><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title1">Best Interaction with the Parts Registry</p><br />
<p>&nbsp;&nbsp;</p><br />
<p><strong>Propose an alternate interaction with the registry. Clearly document this on your wiki in a page explicitly dedicated to this aspect of your tool. You should clearly show how to use this tool in a design flow used by experimental iGEM teams.</strong></p><br />
<p>&nbsp;&nbsp; Biosearch is an iPhone app for partsregistry.org, and has all parts information of Partsregistry database with enhanced user-friendly interface.It has a powerful search engine, users can search parts and devices with keyword, chassis and other types. </p><br />
<p>&nbsp;&nbsp;To see the quick tutorial of Biosearch, please click <a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/Biosearch_Tutorial">tutorial</a>.</p><br />
<p>&nbsp;</p><br />
<p class="title1">&nbsp;&nbsp;Bronze Medal</p><br />
</br><br />
<p><strong>Register the team, have a great summer, and plan to have fun at the Jamboree.</strong></p><br />
<p>&nbsp;&nbsp;We registered our team as SUSTC-Shenzhen-A,enjoyed our great summer and autumn , and we're planning to having fun at the Asia Jamboree!</p><br />
<p>&nbsp;&nbsp;</p><br />
<p><strong>Successfully complete and submit this iGEM 2012 Judging form.</strong></p><br />
<p>&nbsp;&nbsp;To see our judging form, please click <a href="https://igem.org/2012_Judging_Form?id=767">here</a>.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p><strong>Create and share a Description of the team's project using the iGEM wiki.</strong></p><br />
<p>&nbsp;&nbsp;To see our overview of our first project (Biosearch) and second project(Biodesign),please click <a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/Project">here</a>.</p><br />
<p>&nbsp;&nbsp;To see their abstracts:</p><br />
<p>&nbsp;&nbsp; Please click <a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/Human_practice/Project">here</a> for Biosearch.</p> <br />
<p>&nbsp;&nbsp; Please click <a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/Biodesign">here</a> for Biodesign.</p> <br />
<p>&nbsp;&nbsp;</p><br />
<p><strong>Develop and make available via The Registry of Software Tools an open source software tool that supports synthetic biology based on BioBrick standard biological parts.</strong></p><br />
<p>&nbsp;&nbsp; To see our download page of Biosearch, please click <a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/Download">here</a>.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br />
<p class="title1">&nbsp;&nbsp;Sliver Medal</p><br />
</br><br />
<p><strong>Provide a detailed, draft specification for the next version of your software tool.</strong></p><br />
<p>&nbsp;&nbsp;Our next version of Biosearch is in the future plan. Please click <a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/Biosearch_Update">here</a> for more details.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p><strong>Provide a second, distinct software tools project.</strong></p><br />
<p>&nbsp;&nbsp; Our second software is Biodesign, a distinct iPhone app which enables you draft the cell in your pocket!.Please click <a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/Biodesign">here</a> for details!</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title1">&nbsp;&nbsp;Gold Medal</p><br />
</br><br />
<p><strong>Have another team utilize the software developed by your team.</strong></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Eight</strong> iGEM teams utilized our software in their projects, including Tianjin, SJTU-BioX-Shanghai, OUC-China,Tsinghua ,HKUST ,SUSTC-Shenzhen-B, SUSTC-Shenzhen-A. They gave us a number of detailed user experiences and valuable comments. For more details, please click <a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/User_Review">User Review</a>.</p><br />
<p>&nbsp;&nbsp;</p><br />
<br />
<p><strong>Outline and detail how your project effects Human Practice in Synthetic Biology.Such topics include: safety, security, ethics, or ownership, sharing, and innovation.</strong></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Safety issues</strong> (including safety, security, ethics,etc), as a team which devoted ourselves to iPhone app development, we didn’t create any lab product. For more information , please click <a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/Safety">Safety</a>.</p><br />
<p>&nbsp;&nbsp;<strong>Sharing: </strong>With our Biosearch on your iPhone, you can check Biobricks and partsregistry in the seminar room; You can design your genetic circuits when you are waiting for a bus! Biosearch created a user friendly interface and wide using range, enable users to search Biobricks with high efficiency almost everywhere! With local database, users can use Biosearch without connecting internet, making the information of synthetic biology become much easier to get!</p><br />
<p>&nbsp;&nbsp;<strong>Innovation:</strong> Our Biosearch creatively shows and confirms that iPhone can be, and will be an important tool in biology research for its portability. In our human practice, we aim to investigate how an iPhone aids biologists in the lab.</p><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;Want to know more about how our Biosearch effects Human Practice in Synthetic Biology, please click <a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/iphone_biology">here</a>.</p><br />
<p>&nbsp;&nbsp;</p><br />
<br />
<br />
<br />
<p><strong>Develop a new standard, develop and document a new technical standard that supports the sharing BioBrick Parts or Devices, either via physical DNA, or as information via the Internet.</strong></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;We developed a new technical standard: BBF RFC 89 which described the minimum information that a qualified BioBrick should have. There was a BBF RFC 52 before, but as the database is getting larger and larger, a more detailed description is in urgent need.Our RFC 89 will replace the former RFC 52, because it is more precise, and it will surely help to standardize the development of BioBricks. The details can be found in <a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/RFC_draft">RFC draft</a>.</p><br />
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[[file:footbar.jpg|center]]</div>M.B.ZHOUhttp://2012.igem.org/Team:SUSTC-Shenzhen-A/ProjectTeam:SUSTC-Shenzhen-A/Project2012-10-27T03:45:34Z<p>M.B.ZHOU: </p>
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<h1 class="title"><strong>Project Overview</strong></h1><br />
<p class="title1">&nbsp;&nbsp;Biosearch</p><br />
<p><img src="https://static.igem.org/mediawiki/2012/e/e4/BiosearchPage1.png" valign="top" align="right" width="100" style="margin:10px;" /></p><br />
<p>&nbsp;&nbsp;The era of Partsregistry on mobile phone has arrived! With Biosearch on your iPhone, you can now check biobricks and partsregistry in the seminar room; You can design your genetic circuits when you are waiting for a bus! Biosearch is fully interacting with Partsregistry, <a href="http://partsregistry.org">http://partsregistry.org</a>, and has all parts information of Partsregistry database with enhanced user-friendly interface.</p><br />
<p>&nbsp;&nbsp;Biosearch has a powerful search engine. Users can search parts and devices by type, by category, by keywords, etc. Our online survey shows that Biosearch has major improvement in search result ranking. In addition, our iPhone App has new functions including sharing, rating, adding bookmarks and downloading to local system. These new functions shall promote the communicating and sharing between synthetic biologists. The Biosearch is going to be available on Apple Store and is free to use. </p><br />
<p>&nbsp;&nbsp;For more information, please click <a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/Human_practice/Project">here</a>.</p><br />
<br/><br />
<p class="title1">&nbsp;&nbsp;Biodesign</p><br />
<p><img src="https://static.igem.org/mediawiki/2012/a/a6/Sustc_shenzhen_a_biodesign_overview.png" valign="top" align="right" width="100" style="margin:10px;" /></p><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;Our second project, Biodesign, is a graphics editing application specific to synthetic biology for both iPhone and iPad. Biodesign is based on previous iGEM software named Tinkercell and was elaborately improved to adapt to mobile platform. It provides hundreds of biobrick icons as well as lots of fancy functions including dragging graphics, drawing curves and automatically connecting figures with corresponding arrows. With Biosearch, users can draw biochemical reaction schematics anywhere they want and share their drawings with friends. Biodesign will be available online very soon!</p><br />
<p>&nbsp;&nbsp;For more information, please click <a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/Biodesign">here</a>.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title1">&nbsp;&nbsp;RFC</p><br />
<p>&nbsp;&nbsp;We found that the information of many BioBricks is not complete. Thus it is very inconvenient for users to use those information-lacked BioBricks. We want to standardize the minimum information for a qualified BioBrick. If every BioBrick has the same format of information, then it would be very easy to find and distinguish a BioBrick among the sea of BioBricks. In addition, in the website of partsregistry, it is very difficult to find the BioBricks we want due to the useless information in the website. So we conclude that it is essential to create a default template for storing the information of BioBricks. </p><br />
<p>&nbsp;&nbsp;To see a detailed discussion about this BBF RFC draf, please click <a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/RFC">here</a>.</p><br />
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<h1 class="title"><strong>Sponsors</strong></h1><br />
<p>&nbsp;&nbsp;</p><br />
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<td><a href="http://www.sustc.edu.cn"><img src="https://static.igem.org/mediawiki/2012/2/23/Sustc_sponsor_1.png" width="480" height="80" align="left"/></td><br />
<td><p>South University of Science and Technology of China Education Fundation</p></td><br />
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<td><a href="http://www.crcgas.com"><img src="https://static.igem.org/mediawiki/2012/1/10/Sustc_sponsor_2.jpg" width = "280" height="180"align="left"/></td><br />
<td><p>Huarun Gas company</p></td><br />
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<p>&nbsp;&nbsp;We appreciate helps from many faculties and staffers in SUSTC.</p><br />
<p>&nbsp;&nbsp;They are Qinshi Zhu, Qin Ye, Wei Han, xuexian wu, Lingjun Wang, Cuiqiong Pang, Jing Zhang, Yun Jiao, Chaoyang Zhu, Yan Zeng and <br />
many others.</p><br />
<p><strong>&nbsp;&nbsp;Your support is our driving force!</strong><br />
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<h1 class="title"><strong>Acknowledgment</strong></h1><br />
<p>&nbsp;&nbsp;</p><br />
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<td><a href="https://2012.igem.org/Team:HKUST-Hong_Kong"><img src="https://static.igem.org/mediawiki/2012/1/14/HKUST.png" width="300" height="100" align="left"/></td><br />
<td><p>We genuinely appreciate iGEM team from Hong Kong University of Science and Technology to have academic communication with us and provide us with precious suggestions and advice.</p></td><br />
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<td><a href="https://2012.igem.org/Team:Shenzhen"><img src="https://static.igem.org/mediawiki/2012/7/7e/BGI.png" width="300" height="100"align="left"/></td><br />
<td><p>We equally genuinely appreciate iGEM team from BGI to have academic communication with us several times and provide us with precious suggestions and advice.Here we express our thanks especially to Kangkang.</p></td><br />
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<td><a href="https://2012.igem.org/Team:Tsinghua"><img src="https://static.igem.org/mediawiki/2012/a/ae/Tsinghua.png" width="300" height="100"align="left"/></td><br />
<td><p>We would also like to give our thanks to iGEM team from Tsinghua University who participate in our survey and give us remarks which truly help us make improvements in our iPhone App.</p></td><br />
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<td><a href="https://2012.igem.org/Team:OUC-China"><img src="https://static.igem.org/mediawiki/2012/d/dd/OUC.png" width="300" height="100"align="left"/></td><br />
<td><p>Thanks to iGEM team from Ocean University of China, we received very good user review about our iPhone App.</p></td><br />
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<td><a href="https://2012.igem.org/Team:Tianjin"><img src="https://static.igem.org/mediawiki/2012/b/be/Tianjin.png" width="300" height="100" align="left"/></td><br />
<td><p>iGEM team from Tianjin University have participated in our survey and send us pieces of constructive advice.Thank you all.</p></td><br />
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<td><a href="https://2012.igem.org/Team:SUSTC-Shenzhen-B"><img src="https://static.igem.org/mediawiki/2012/1/16/Photo_WIKI2.png" width="300" height="100" align="left"/></td><br />
<td><p>Here we give our special thanks to team SUSTC-Shenzhen-B. We always grow hand in hand, shoulder by shoulder. We hope both of us can achieve what we deserve. </p></td><br />
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[[file:footbar.jpg|center]]</div>M.B.ZHOUhttp://2012.igem.org/File:Sustc_shenzhen_a_biodesign_overview.pngFile:Sustc shenzhen a biodesign overview.png2012-10-27T03:44:19Z<p>M.B.ZHOU: </p>
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<div></div>M.B.ZHOUhttp://2012.igem.org/Team:SUSTC-Shenzhen-A/User_ReviewTeam:SUSTC-Shenzhen-A/User Review2012-10-27T03:43:22Z<p>M.B.ZHOU: </p>
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<p>&nbsp;Our software is very popular among iGEM teams and synthetic biologists. There are more than 20 users in less than one week time after we released Beta version of the software in Asia. The user distribution in Asia is shown in the map. We did a survey about comments on Partsregistry and our software. The result is listed below.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title1">&nbsp;&nbsp;Users Around the World</p><br />
<p>&nbsp;&nbsp;Since the time our BioSearch has been launched on APP Store, our clients around the world had used this software!<br/><br />
The figure below shows the user distribution. Green place indicates the country that the user locates.</p><br />
<p>&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2012/c/c3/Sustc_shenzhen_a_Wordmap.png"align="center"></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;In order to satisfy the need of major iGEM participants and synthetic biologists, SUSTC-Shenzhen-A send a survey to several iGEM teams in mainland China to collect their opinions on Partsregistry and our App. You can see from the following figure, teams from <B><I>Ocean University of China, Shanghai Jiaotong University, Tianjin University and Tsinghua University</B></I> are very active in our surveys.They highly think of our project and provide us with a lot of precious suggestions. Here we want to express our appriciation to them from the deep heart.</p><br />
<p>&nbsp;&nbsp;</p><br />
<img src="https://static.igem.org/mediawiki/2012/8/8e/SUSTC_UR1.jpg"width=440><br />
<img src="https://static.igem.org/mediawiki/2012/9/9b/SUSTC_UR2.jpg"width=440><br />
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<p>&nbsp;&nbsp;</p><br />
<p><strong>&nbsp;&nbsp;&nbsp;&nbsp;Question1: Registry of standard biological parts(http://partsregistry.org) provides users with a search bar on the top of the right corner. Are you satisfied with its searching result?</strong></p><br />
<p><strong>&nbsp;&nbsp;&nbsp;&nbsp;Question2: Will you expect us to make Partsregistry an iPhone App?</strong></p><br />
<br><br />
<br />
<br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;The figure shows that 8% users feel unsatisfactory about Partsregistry, 46% feel acceptable, 46% feel satisfactory about it.Therefore, it's our responsibility to make it more convenient for researchers to search for their desired biobricks. Another survey shows that over 85% of iGEM participants are in an urgent need for an iPhone App about Partsregistry. That strongly demonstrate the significance to proceed this project. </p><br />
<br><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/6/62/SUSTC_UR3.jpg"width=440><br />
<img src="https://static.igem.org/mediawiki/2012/5/5c/SUSTC_UR4.jpg"width=440><br />
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<p><strong>&nbsp;&nbsp;&nbsp;&nbsp;Question3: How do you evaluate the search function in BioSearch according to your firsthand user experience?<br />
</strong></p><br />
<p><strong>&nbsp;&nbsp;&nbsp;&nbsp;Question4:How do you think of our effort in optimizing page layout and human oriented design?<br />
</strong></p><br />
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<p>&nbsp;&nbsp;&nbsp;&nbsp;The figure shows that more than 60% of the users think our software is both functional and beautiful.</p><br />
<br><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title1">&nbsp;&nbsp;User Comment</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>austinamens </strong>from Tianjin</p><br />
<p>&nbsp;&nbsp;I searched by keywords and by part name. BioSearch is indeed a good trying.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong> jiaquan_good </strong>from Tianjin.</p><br />
<p>&nbsp;&nbsp;I looked over bioparts list by category. It is easy to use and the tutorial is very helpful.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>huanan1991 </strong>from SJTU-BioX-Shanghai</p><br />
<p>&nbsp;&nbsp;I searched and viewed several lists by catalog. I use BioaSearch to search bioparts relative to synthesis of fatty acid.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Adjaniyu</strong> from SJTU-BioX-Shanghai</p><br />
<p>&nbsp;&nbsp;I’m very excited to see such a pithy user interface. I searched several vio-operon parts.<p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Vampire19</strong> from OUC-iGEM</p><br />
<p>&nbsp;&nbsp;I searched BioBrick J23015 and clicked the link in BioSearch to view experience page on website.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Oucigem1</strong> from OUC-China</p><br />
<p>&nbsp;&nbsp;The overall effect of BioSearch is good. I searched BBa_K116401 and BBa_J23101.<p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Newfive </strong>from OUC-China<p><br />
<p>&nbsp;&nbsp;With BioSearch, I searched BBa_K737005 and added it to “Favorite”.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Pengyongouc </strong>from OUC-China</p><br />
<p>&nbsp;&nbsp;I searched Bba_K737067 and got expecting result.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>cwth166</strong> from OUC-China</p><br />
<p>&nbsp;&nbsp;I searched J23106, R0011 and E0480. It is fast and accurate, and it’s very beautiful.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Oucigem2 </strong> from OUC-China</p><br />
<p>&nbsp;&nbsp;As for its search function, BioSearch is strong and accurate, though it is not a multi-function. I searched BBa-J23106. </p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>Liuerlrir </strong> from OUC-China</p><br />
<p>&nbsp;&nbsp;I tried all the function of BioSearch and searched BBa_R0082, cph8 and k081024. I thought I got what I wanted.</p><br />
<p>&nbsp;&nbsp;<p><br />
<p>&nbsp;&nbsp;<strong>530854106QQ</strong> from OUC-China</p><br />
<p>&nbsp;&nbsp;I searched Bba_K737067 with BioSearch on iPhone which is much faster than Partsregistry. I really expect you to develop an iPad version.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>kaykang510</strong> from Tsinghua</p><br />
<p>&nbsp;&nbsp;I tried search by name, by keywords, by type and by catalog. I scanned sub list of LuxI, LuxR, Las and FemE.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;<strong>astrol.lee</strong> from Tsinghua</p><br />
<p>&nbsp;&nbsp;I viewed the lists of RFP, cI and lacI. It’s fast and accurate.</p><br />
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[[file:footbar.jpg|center]]</div>M.B.ZHOUhttp://2012.igem.org/File:Sustc_shenzhen_a_Wordmap.pngFile:Sustc shenzhen a Wordmap.png2012-10-27T03:39:17Z<p>M.B.ZHOU: </p>
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<div></div>M.B.ZHOUhttp://2012.igem.org/File:SUSTC_map3.jpgFile:SUSTC map3.jpg2012-10-27T03:37:21Z<p>M.B.ZHOU: uploaded a new version of &quot;File:SUSTC map3.jpg&quot;</p>
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<div></div>M.B.ZHOUhttp://2012.igem.org/Team:SUSTC-Shenzhen-A/Biodesign_TutorialTeam:SUSTC-Shenzhen-A/Biodesign Tutorial2012-10-27T03:33:57Z<p>M.B.ZHOU: </p>
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<td><div><br />
<br />
<h1 class="title">&nbsp;BioDesign Tutorial</h1><br />
<br />
<br />
<a name="Video" ></a> <br />
<h3 class="title1">&nbsp;&nbsp;Video tutorial</h3><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/Devidingline_whole.jpg"/><br />
<p>&nbsp;&nbsp;Note: There will be a 6-second Chinese ad at the beginning of this video.</p><br />
<p align="center"><br />
<embed src="http://player.youku.com/player.php/sid/XNDY3Mzg4Njcy/v.swf" <br />
width="480" height="400" <br />
type="application/x-shockwave-flash"><br />
</embed></p><br />
<br />
<h3 class="title1">&nbsp;&nbsp;Step by step instruction</h3><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/Devidingline_whole.jpg"/><br />
<ul><br />
<a href="#Folder">Folder page <br />
</a> &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
<br />
<a href="#File">File page <br />
</a>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
<br />
<a href="#Drawing">Drawing page <br />
</a>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
<br />
<a href="#Mail">Mail page <br />
</a> &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
<br />
</ul><br />
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<td>&nbsp;</td><br />
</tr><br />
<br />
<br />
<tr><td><a name="Folder" ></a> <br />
<p class="title1">&nbsp;&nbsp;Folder page</p><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/Devidingline_whole.jpg"/><br />
<br />
<p><img src="https://static.igem.org/mediawiki/2012/e/e6/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_1.png" valign="top" align="left" width="300" height = "480" style="BORDER:#CCFFCC 5px dashed;margin:10px;" ><img src="https://static.igem.org/mediawiki/2012/b/b7/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_2.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:10px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(1)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Fig.1 is the first page. </p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;In order to create a new project/folder, you should click the ‘add’ button at the top right (Fig.2).</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(2)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
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<p><img src="https://static.igem.org/mediawiki/2012/3/35/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_3.png" valign="top" align="left" height="480" width="300" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(3)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the icon, then click ‘enter’,<br/> it goes into the secondary page (Fig.3).</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
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<p>&nbsp;&nbsp;</p><br />
<br />
</td><br />
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<br />
<tr><br />
<td><a name="File" ></a> <br />
<p class="title1">&nbsp;&nbsp;File page</p><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/Devidingline_whole.jpg"/><br />
<p><img src="https://static.igem.org/mediawiki/2012/2/26/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_4.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/6/60/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_5.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:10px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(4)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;The secondary page looks like Fig.4. </p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the button name ‘add’ at the top right to create a new file (Fig.5).</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(5)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
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<br />
<tr><br />
<td><br />
<a name="Drawing"></a><br />
<p class="title1">&nbsp;&nbsp;Drawing page</p><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/Devidingline_whole.jpg"/><br />
<p><img src="https://static.igem.org/mediawiki/2012/4/41/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_6.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/5/54/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_7.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(6)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the newly created file, choose ‘edit’ to do the drawing (Fig.6).</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click a object at the bottom to set it on the screen.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(7)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
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<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/8/8f/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_8.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/b/b2/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_9.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(8)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the text field next to a part to rename this part.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;For the objects in “comp”, you can zoom them by ywo fingers to change the their sizes.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(9)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/1/14/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_10.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/f/f6/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_11.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(10)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can rotate the object clockwisely by clicking the rotating button.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can get a vector on the canvas from “part”.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(11)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
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<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/f/fd/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_12.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/c/c8/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_13.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(12)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can change the size of the vector by zooming with two fingers.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;If you have add a cell, you may find it covers the vector or other object. Click the “down” button to lower layer.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(13)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
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<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/6/63/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_14.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/7/71/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_15.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(14)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can put objects(in”mole. & part.”) on vector by draging it into the vector.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;In “reac.”, you have various choices to connect objects by Bezier curve.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(15)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
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<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/2/24/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_16.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/f/fa/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_17.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(16)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click two objects in turn and they will be connected.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can click the place near the line to pitch the curve. And then change the shape of the curve by dragging with one or two fingers.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(17)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
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<br/><br />
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<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/c/cb/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_18.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/9/99/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_19.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(18)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can change the type of the arrow by clicking the first button at the right.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Fig.19 shows the changed arrow.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(19)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/7/77/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_20.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/0/0e/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_21.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(20)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can pitch the center point to edit both parts at the same time.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;In “regu.”, choose one to connect two or three parts by polygonal line.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(21)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/c/ca/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_22.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/0/00/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_23.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(22)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Fig.22 shows what happens after choosing the "regu".</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Fig.23 also shows what happens after choosing the "regu".</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(23)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/6/66/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_24.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/6/6d/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_25.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(24)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;For objects in “part.”, you can move one group close to another to connet them.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can press the button to disconnect them.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(25)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/d/df/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_70.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/9/90/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_71.png" valign="top" align="right" width="300"height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(26)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the button at the top right to save this picture (Fig.26). </p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Come back, you’ll see the icon has change into the saved picture (Fig.27).</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(27)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/e/ef/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_72.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/f/f8/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_73.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(28)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the microscope button in FIle page at the bottom to search a file (Fig.28). </p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbspClick the button at the bottom right to present the files in a table (Fig.29).</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(29)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><a name="Mail" ></a> <br />
<p class="title1">&nbsp;&nbsp;Mail page</p><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/Devidingline_whole.jpg" /><br />
<p><img src="https://static.igem.org/mediawiki/2012/b/bf/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_74.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/5/5a/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_75.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:10px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(30)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Come back to the Folder page. Click the envelope button at the bottom right to the file that you want to email to your friend! (Fig.30)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the ‘done’ button. Send the chosen files to your friends (Fig.31)!</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(31)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<br />
<br />
</table><br />
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<div></td><br />
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[[file:footbar.jpg|center]]</div>M.B.ZHOUhttp://2012.igem.org/Team:SUSTC-Shenzhen-B/protocol1Team:SUSTC-Shenzhen-B/protocol12012-10-26T21:47:23Z<p>M.B.ZHOU: </p>
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no-repeat;cursor:pointer}div.light_rounded .pp_expand{background:url(../images/light_rounded/sprite.png) -31px -26px no-repeat;cursor:pointer}div.light_rounded .pp_expand:hover{background:url(../images/light_rounded/sprite.png) -31px -47px no-repeat;cursor:pointer}div.light_rounded .pp_contract{background:url(../images/light_rounded/sprite.png) 0 -26px no-repeat;cursor:pointer}div.light_rounded .pp_contract:hover{background:url(../images/light_rounded/sprite.png) 0 -47px no-repeat;cursor:pointer}div.light_rounded .pp_close{background:url(../images/light_rounded/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:75px}div.light_rounded .pp_nav .pp_play{background:url(../images/light_rounded/sprite.png) -1px -100px no-repeat;height:15px;width:14px}div.light_rounded .pp_nav .pp_pause{background:url(../images/light_rounded/sprite.png) -24px -100px no-repeat;height:15px;width:14px}div.light_rounded .pp_arrow_previous{background:url(../images/light_rounded/sprite.png) 0 -71px no-repeat}div.light_rounded .pp_arrow_next{background:url(../images/light_rounded/sprite.png) -22px -71px no-repeat}div.light_rounded .pp_bottom .pp_left{background:url(../images/light_rounded/sprite.png) -88px -80px no-repeat}div.light_rounded .pp_bottom .pp_right{background:url(../images/light_rounded/sprite.png) -110px -80px no-repeat}div.dark_rounded .pp_top .pp_left{background:url(../images/dark_rounded/sprite.png) -88px -53px no-repeat}div.dark_rounded .pp_top .pp_right{background:url(../images/dark_rounded/sprite.png) -110px -53px no-repeat}div.dark_rounded .pp_content_container .pp_left{background:url(../images/dark_rounded/contentPattern.png) top left repeat-y}div.dark_rounded .pp_content_container .pp_right{background:url(../images/dark_rounded/contentPattern.png) top right repeat-y}div.dark_rounded .pp_next:hover{background:url(../images/dark_rounded/btnNext.png) center right no-repeat;cursor:pointer}div.dark_rounded .pp_previous:hover{background:url(../images/dark_rounded/btnPrevious.png) center left no-repeat;cursor:pointer}div.dark_rounded .pp_expand{background:url(../images/dark_rounded/sprite.png) -31px -26px no-repeat;cursor:pointer}div.dark_rounded .pp_expand:hover{background:url(../images/dark_rounded/sprite.png) -31px -47px no-repeat;cursor:pointer}div.dark_rounded .pp_contract{background:url(../images/dark_rounded/sprite.png) 0 -26px no-repeat;cursor:pointer}div.dark_rounded .pp_contract:hover{background:url(../images/dark_rounded/sprite.png) 0 -47px no-repeat;cursor:pointer}div.dark_rounded .pp_close{background:url(../images/dark_rounded/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:75px}div.dark_rounded .pp_description{color:#fff;margin-right:85px}div.dark_rounded .pp_nav .pp_play{background:url(../images/dark_rounded/sprite.png) -1px -100px no-repeat;height:15px;width:14px}div.dark_rounded .pp_nav .pp_pause{background:url(../images/dark_rounded/sprite.png) -24px -100px no-repeat;height:15px;width:14px}div.dark_rounded .pp_arrow_previous{background:url(../images/dark_rounded/sprite.png) 0 -71px no-repeat}div.dark_rounded .pp_arrow_next{background:url(../images/dark_rounded/sprite.png) -22px -71px no-repeat}div.dark_rounded .pp_bottom .pp_left{background:url(../images/dark_rounded/sprite.png) -88px -80px no-repeat}div.dark_rounded .pp_bottom .pp_right{background:url(../images/dark_rounded/sprite.png) -110px -80px no-repeat}div.dark_rounded .pp_loaderIcon{background:url(../images/dark_rounded/loader.gif) center center no-repeat}div.dark_square .pp_left,div.dark_square .pp_middle,div.dark_square .pp_right,div.dark_square .pp_content{background:#000}div.dark_square .pp_description{color:#fff;margin:0 85px 0 0}div.dark_square .pp_loaderIcon{background:url(../images/dark_square/loader.gif) center center no-repeat}div.dark_square .pp_expand{background:url(../images/dark_square/sprite.png) -31px -26px no-repeat;cursor:pointer}div.dark_square .pp_expand:hover{background:url(../images/dark_square/sprite.png) -31px -47px no-repeat;cursor:pointer}div.dark_square .pp_contract{background:url(../images/dark_square/sprite.png) 0 -26px no-repeat;cursor:pointer}div.dark_square .pp_contract:hover{background:url(../images/dark_square/sprite.png) 0 -47px no-repeat;cursor:pointer}div.dark_square .pp_close{background:url(../images/dark_square/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:75px}div.dark_square .pp_nav{clear:none}div.dark_square .pp_nav .pp_play{background:url(../images/dark_square/sprite.png) -1px -100px no-repeat;height:15px;width:14px}div.dark_square .pp_nav .pp_pause{background:url(../images/dark_square/sprite.png) -24px -100px no-repeat;height:15px;width:14px}div.dark_square .pp_arrow_previous{background:url(../images/dark_square/sprite.png) 0 -71px no-repeat}div.dark_square .pp_arrow_next{background:url(../images/dark_square/sprite.png) -22px -71px no-repeat}div.dark_square .pp_next:hover{background:url(../images/dark_square/btnNext.png) center right no-repeat;cursor:pointer}div.dark_square .pp_previous:hover{background:url(../images/dark_square/btnPrevious.png) center left no-repeat;cursor:pointer}div.light_square .pp_expand{background:url(../images/light_square/sprite.png) -31px -26px no-repeat;cursor:pointer}div.light_square .pp_expand:hover{background:url(../images/light_square/sprite.png) -31px -47px no-repeat;cursor:pointer}div.light_square .pp_contract{background:url(../images/light_square/sprite.png) 0 -26px no-repeat;cursor:pointer}div.light_square .pp_contract:hover{background:url(../images/light_square/sprite.png) 0 -47px no-repeat;cursor:pointer}div.light_square .pp_close{background:url(../images/light_square/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:75px}div.light_square .pp_nav .pp_play{background:url(../images/light_square/sprite.png) -1px -100px no-repeat;height:15px;width:14px}div.light_square .pp_nav .pp_pause{background:url(../images/light_square/sprite.png) -24px -100px no-repeat;height:15px;width:14px}div.light_square .pp_arrow_previous{background:url(../images/light_square/sprite.png) 0 -71px no-repeat}div.light_square .pp_arrow_next{background:url(../images/light_square/sprite.png) -22px -71px no-repeat}div.light_square .pp_next:hover{background:url(../images/light_square/btnNext.png) center right no-repeat;cursor:pointer}div.light_square .pp_previous:hover{background:url(../images/light_square/btnPrevious.png) center left no-repeat;cursor:pointer}div.facebook .pp_top .pp_left{background:url(../images/facebook/sprite.png) -88px -53px no-repeat}div.facebook .pp_top .pp_middle{background:url(../images/facebook/contentPatternTop.png) top left repeat-x}div.facebook .pp_top .pp_right{background:url(../images/facebook/sprite.png) -110px -53px no-repeat}div.facebook .pp_content_container .pp_left{background:url(../images/facebook/contentPatternLeft.png) top left repeat-y}div.facebook .pp_content_container .pp_right{background:url(../images/facebook/contentPatternRight.png) top right repeat-y}div.facebook .pp_expand{background:url(../images/facebook/sprite.png) -31px -26px no-repeat;cursor:pointer}div.facebook .pp_expand:hover{background:url(../images/facebook/sprite.png) -31px -47px no-repeat;cursor:pointer}div.facebook .pp_contract{background:url(../images/facebook/sprite.png) 0 -26px no-repeat;cursor:pointer}div.facebook .pp_contract:hover{background:url(../images/facebook/sprite.png) 0 -47px no-repeat;cursor:pointer}div.facebook .pp_close{background:url(../images/facebook/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:22px}div.facebook .pp_description{margin:0 37px 0 0}div.facebook .pp_loaderIcon{background:url(../images/facebook/loader.gif) center center no-repeat}div.facebook .pp_arrow_previous{background:url(../images/facebook/sprite.png) 0 -71px no-repeat;height:22px;margin-top:0;width:22px}div.facebook .pp_arrow_previous.disabled{background-position:0 -96px;cursor:default}div.facebook .pp_arrow_next{background:url(../images/facebook/sprite.png) -32px -71px no-repeat;height:22px;margin-top:0;width:22px}div.facebook .pp_arrow_next.disabled{background-position:-32px -96px;cursor:default}div.facebook .pp_nav{margin-top:0}div.facebook .pp_nav p{font-size:15px;padding:0 3px 0 4px}div.facebook .pp_nav .pp_play{background:url(../images/facebook/sprite.png) -1px -123px no-repeat;height:22px;width:22px}div.facebook .pp_nav .pp_pause{background:url(../images/facebook/sprite.png) -32px -123px no-repeat;height:22px;width:22px}div.facebook .pp_next:hover{background:url(../images/facebook/btnNext.png) center right no-repeat;cursor:pointer}div.facebook .pp_previous:hover{background:url(../images/facebook/btnPrevious.png) center left no-repeat;cursor:pointer}div.facebook .pp_bottom .pp_left{background:url(../images/facebook/sprite.png) -88px -80px no-repeat}div.facebook .pp_bottom .pp_middle{background:url(../images/facebook/contentPatternBottom.png) top left repeat-x}div.facebook .pp_bottom .pp_right{background:url(../images/facebook/sprite.png) -110px -80px no-repeat}div.pp_pic_holder a:focus{outline:0}div.pp_overlay{background:#000;display:none;left:0;position:absolute;top:0;width:100%;z-index:9500}div.pp_pic_holder{display:none;position:absolute;width:100px;z-index:10000}.pp_content{height:40px;min-width:40px}* html .pp_content{width:40px}.pp_content_container{position:relative;text-align:left;width:100%}.pp_content_container .pp_left{padding-left:20px}.pp_content_container .pp_right{padding-right:20px}.pp_content_container .pp_details{float:left;margin:10px 0 2px}.pp_description{display:none;margin:0}.pp_social{float:left;margin:0}.pp_social .facebook{float:left;margin-left:5px;overflow:hidden;width:55px}.pp_social .twitter{float:left}.pp_nav{clear:right;float:left;margin:3px 10px 0 0}.pp_nav p{float:left;margin:2px 4px;white-space:nowrap}.pp_nav .pp_play,.pp_nav .pp_pause{float:left;margin-right:4px;text-indent:-10000px}a.pp_arrow_previous,a.pp_arrow_next{display:block;float:left;height:15px;margin-top:3px;overflow:hidden;text-indent:-10000px;width:14px}.pp_hoverContainer{position:absolute;top:0;width:100%;z-index:2000}.pp_gallery{display:none;left:50%;margin-top:-50px;position:absolute;z-index:10000}.pp_gallery div{float:left;overflow:hidden;position:relative}.pp_gallery ul{float:left;height:35px;margin:0 0 0 5px;padding:0;position:relative;white-space:nowrap}.pp_gallery ul a{border:1px rgba(0,0,0,.5) solid;display:block;float:left;height:33px;overflow:hidden}.pp_gallery ul a img{border:0}.pp_gallery li{display:block;float:left;margin:0 5px 0 0;padding:0}.pp_gallery li.default a{background:url(../images/facebook/default_thumbnail.gif) 0 0 no-repeat;display:block;height:33px;width:50px}.pp_gallery .pp_arrow_previous,.pp_gallery .pp_arrow_next{margin-top:7px!important}a.pp_next{background:url(../images/light_rounded/btnNext.png) 10000px 10000px no-repeat;display:block;float:right;height:100%;text-indent:-10000px;width:49%}a.pp_previous{background:url(../images/light_rounded/btnNext.png) 10000px 10000px no-repeat;display:block;float:left;height:100%;text-indent:-10000px;width:49%}a.pp_expand,a.pp_contract{cursor:pointer;display:none;height:20px;position:absolute;right:30px;text-indent:-10000px;top:10px;width:20px;z-index:20000}a.pp_close{display:block;line-height:22px;position:absolute;right:0;text-indent:-10000px;top:0}.pp_loaderIcon{display:block;height:24px;left:50%;margin:-12px 0 0 -12px;position:absolute;top:50%;width:24px}#pp_full_res{line-height:1!important}#pp_full_res .pp_inline{text-align:left}#pp_full_res .pp_inline p{margin:0 0 15px}div.ppt{color:#fff;display:none;font-size:17px;margin:0 0 5px 15px;z-index:9999}div.pp_default .pp_content,div.light_rounded .pp_content{background-color:#fff}div.pp_default #pp_full_res .pp_inline,div.light_rounded .pp_content .ppt,div.light_rounded #pp_full_res .pp_inline,div.light_square .pp_content .ppt,div.light_square #pp_full_res .pp_inline,div.facebook .pp_content .ppt,div.facebook #pp_full_res .pp_inline{color:#000}div.pp_default .pp_gallery ul li a:hover,div.pp_default .pp_gallery ul li.selected a,.pp_gallery ul a:hover,.pp_gallery li.selected a{border-color:#fff}div.pp_default .pp_details,div.light_rounded .pp_details,div.dark_rounded .pp_details,div.dark_square .pp_details,div.light_square .pp_details,div.facebook .pp_details{position:relative}div.light_rounded .pp_top .pp_middle,div.light_rounded .pp_content_container .pp_left,div.light_rounded .pp_content_container .pp_right,div.light_rounded .pp_bottom .pp_middle,div.light_square .pp_left,div.light_square .pp_middle,div.light_square .pp_right,div.light_square .pp_content,div.facebook .pp_content{background:#fff}div.light_rounded .pp_description,div.light_square .pp_description{margin-right:85px}div.light_rounded .pp_gallery a.pp_arrow_previous,div.light_rounded .pp_gallery a.pp_arrow_next,div.dark_rounded .pp_gallery a.pp_arrow_previous,div.dark_rounded .pp_gallery a.pp_arrow_next,div.dark_square .pp_gallery a.pp_arrow_previous,div.dark_square .pp_gallery a.pp_arrow_next,div.light_square .pp_gallery a.pp_arrow_previous,div.light_square .pp_gallery a.pp_arrow_next{margin-top:12px!important}div.light_rounded .pp_arrow_previous.disabled,div.dark_rounded .pp_arrow_previous.disabled,div.dark_square .pp_arrow_previous.disabled,div.light_square .pp_arrow_previous.disabled{background-position:0 -87px;cursor:default}div.light_rounded .pp_arrow_next.disabled,div.dark_rounded .pp_arrow_next.disabled,div.dark_square .pp_arrow_next.disabled,div.light_square .pp_arrow_next.disabled{background-position:-22px -87px;cursor:default}div.light_rounded .pp_loaderIcon,div.light_square .pp_loaderIcon{background:url(../images/light_rounded/loader.gif) center center no-repeat}div.dark_rounded .pp_top .pp_middle,div.dark_rounded .pp_content,div.dark_rounded .pp_bottom .pp_middle{background:url(../images/dark_rounded/contentPattern.png) top left repeat}div.dark_rounded .currentTextHolder,div.dark_square .currentTextHolder{color:#c4c4c4}div.dark_rounded #pp_full_res .pp_inline,div.dark_square #pp_full_res .pp_inline{color:#fff}.pp_top,.pp_bottom{height:20px;position:relative}* html .pp_top,* html .pp_bottom{padding:0 20px}.pp_top .pp_left,.pp_bottom .pp_left{height:20px;left:0;position:absolute;width:20px}.pp_top .pp_middle,.pp_bottom .pp_middle{height:20px;left:20px;position:absolute;right:20px}* html .pp_top .pp_middle,* html .pp_bottom .pp_middle{left:0;position:static}.pp_top .pp_right,.pp_bottom .pp_right{height:20px;left:auto;position:absolute;right:0;top:0;width:20px}.pp_fade,.pp_gallery li.default a img{display:none}<br />
<br />
</style><br />
<script type="text/javascript"><br />
<br />
/*<br />
* Superfish v1.4.8 - jQuery menu widget<br />
* Copyright (c) 2008 Joel Birch<br />
*<br />
* Dual licensed under the MIT and GPL licenses:<br />
* http://www.opensource.org/licenses/mit-license.php<br />
* http://www.gnu.org/licenses/gpl.html<br />
*<br />
* CHANGELOG: http://users.tpg.com.au/j_birch/plugins/superfish/changelog.txt<br />
*/<br />
<br />
;(function($){<br />
$.fn.superfish = function(op){<br />
<br />
var sf = $.fn.superfish,<br />
c = sf.c,<br />
$arrow = $(['<span class="',c.arrowClass,'"> &#187;</span>'].join('')),<br />
over = function(){<br />
var $$ = $(this), menu = getMenu($$);<br />
clearTimeout(menu.sfTimer);<br />
$$.showSuperfishUl().siblings().hideSuperfishUl();<br />
},<br />
out = function(){<br />
var $$ = $(this), menu = getMenu($$), o = sf.op;<br />
clearTimeout(menu.sfTimer);<br />
menu.sfTimer=setTimeout(function(){<br />
o.retainPath=($.inArray($$[0],o.$path)>-1);<br />
$$.hideSuperfishUl();<br />
//if (o.$path.length &amp;&amp; $$.parents(['li.',o.hoverClass].join('')).length<1){over.call(o.$path);}<br />
if (o.$path.length) {<br />
if ($$.parents(['li.',o.hoverClass].join('')).length<1){over.call(o.$path);}<br />
}<br />
},o.delay); <br />
},<br />
getMenu = function($menu){<br />
var menu = $menu.parents(['ul.',c.menuClass,':first'].join(''))[0];<br />
sf.op = sf.o[menu.serial];<br />
return menu;<br />
},<br />
addArrow = function($a){ $a.addClass(c.anchorClass).append($arrow.clone()); };<br />
<br />
return this.each(function() {<br />
var s = this.serial = sf.o.length;<br />
var o = $.extend({},sf.defaults,op);<br />
o.$path = $('li.'+o.pathClass,this).slice(0,o.pathLevels).each(function(){<br />
$(this).addClass([o.hoverClass,c.bcClass].join(' '))<br />
.filter('li:has(ul)').removeClass(o.pathClass);<br />
});<br />
sf.o[s] = sf.op = o;<br />
var bcheck = false;<br />
if ($.fn.hoverIntent) {<br />
if (!o.disableHI) bcheck = true;<br />
}<br />
<br />
$('li:has(ul)',this)[(bcheck) ? 'hoverIntent' : 'hover'](over,out).each(function() {<br />
if (o.autoArrows) addArrow( $('>a:first-child',this) );<br />
})<br />
.not('.'+c.bcClass)<br />
.hideSuperfishUl();<br />
<br />
var $a = $('a',this);<br />
$a.each(function(i){<br />
var $li = $a.eq(i).parents('li');<br />
$a.eq(i).focus(function(){over.call($li);}).blur(function(){out.call($li);});<br />
});<br />
o.onInit.call(this);<br />
<br />
}).each(function() {<br />
var menuClasses = [c.menuClass];<br />
//if (sf.op.dropShadows &amp;&amp; !($.browser.msie &amp;&amp; $.browser.version < 7)) menuClasses.push(c.shadowClass);<br />
if (sf.op.dropShadows) if (!$.browser.msie) if (!($.browser.version < 7)) menuClasses.push(c.shadowClass);<br />
$(this).addClass(menuClasses.join(' '));<br />
});<br />
};<br />
<br />
var sf = $.fn.superfish;<br />
sf.o = [];<br />
sf.op = {};<br />
sf.IE7fix = function(){<br />
var o = sf.op;<br />
//if ($.browser.msie &amp;&amp; $.browser.version > 6 &amp;&amp; o.dropShadows &amp;&amp; o.animation.opacity!=undefined)<br />
if ($.browser.msie) if($.browser.version > 6) if (o.dropShadows) if (o.animation.opacity!=undefined)<br />
this.toggleClass(sf.c.shadowClass+'-off');<br />
};<br />
sf.c = {<br />
bcClass : 'sf-breadcrumb',<br />
menuClass : 'sf-js-enabled',<br />
anchorClass : 'sf-with-ul',<br />
arrowClass : 'sf-sub-indicator',<br />
shadowClass : 'sf-shadow'<br />
};<br />
sf.defaults = {<br />
hoverClass : 'sfHover',<br />
pathClass : 'overideThisToUse',<br />
pathLevels : 1,<br />
delay : 800,<br />
animation : {opacity:'show'},<br />
speed : 'normal',<br />
autoArrows : true,<br />
dropShadows : true,<br />
disableHI : false, // true disables hoverIntent detection<br />
onInit : function(){}, // callback functions<br />
onBeforeShow: function(){},<br />
onShow : function(){},<br />
onHide : function(){}<br />
};<br />
$.fn.extend({<br />
hideSuperfishUl : function(){<br />
var o = sf.op,<br />
not = (o.retainPath===true) ? o.$path : '';<br />
o.retainPath = false;<br />
var $ul = $(['li.',o.hoverClass].join(''),this).add(this).not(not).removeClass(o.hoverClass)<br />
.find('>ul').hide().css('visibility','hidden');<br />
o.onHide.call($ul);<br />
return this;<br />
},<br />
showSuperfishUl : function(){<br />
var o = sf.op,<br />
sh = sf.c.shadowClass+'-off',<br />
$ul = this.addClass(o.hoverClass)<br />
.find('>ul:hidden').css('visibility','visible');<br />
sf.IE7fix.call($ul);<br />
o.onBeforeShow.call($ul);<br />
$ul.animate(o.animation,o.speed,function(){ sf.IE7fix.call($ul); o.onShow.call($ul); });<br />
return this;<br />
}<br />
});<br />
<br />
})(jQuery);<br />
<br />
/*<br />
* Supersubs v0.2b - jQuery plugin<br />
* Copyright (c) 2008 Joel Birch<br />
*<br />
* Dual licensed under the MIT and GPL licenses:<br />
* http://www.opensource.org/licenses/mit-license.php<br />
* http://www.gnu.org/licenses/gpl.html<br />
*<br />
*<br />
* This plugin automatically adjusts submenu widths of suckerfish-style menus to that of<br />
* their longest list item children. If you use this, please expect bugs and report them<br />
* to the jQuery Google Group with the word 'Superfish' in the subject line.<br />
*<br />
*/<br />
<br />
(function($){ // $ will refer to jQuery within this closure<br />
<br />
$.fn.supersubs = function(options){<br />
var opts = $.extend({}, $.fn.supersubs.defaults, options);<br />
// return original object to support chaining<br />
return this.each(function() {<br />
// cache selections<br />
var $$ = $(this);<br />
// support metadata<br />
var o = $.meta ? $.extend({}, opts, $$.data()) : opts;<br />
// get the font size of menu.<br />
// .css('fontSize') returns various results cross-browser, so measure an em dash instead<br />
var fontsize = $('<li id="menu-fontsize">&#8212;</li>').css({<br />
'padding' : 0,<br />
'position' : 'absolute',<br />
'top' : '-999em',<br />
'width' : 'auto'<br />
}).appendTo($$).width(); //clientWidth is faster, but was incorrect here<br />
// remove em dash<br />
$('#menu-fontsize').remove();<br />
// cache all ul elements<br />
$ULs = $$.find('ul');<br />
// loop through each ul in menu<br />
$ULs.each(function(i) { <br />
// cache this ul<br />
var $ul = $ULs.eq(i);<br />
// get all (li) children of this ul<br />
var $LIs = $ul.children();<br />
// get all anchor grand-children<br />
var $As = $LIs.children('a');<br />
// force content to one line and save current float property<br />
var liFloat = $LIs.css('white-space','nowrap').css('float');<br />
// remove width restrictions and floats so elements remain vertically stacked<br />
var emWidth = $ul.add($LIs).add($As).css({<br />
'float' : 'none',<br />
'width' : 'auto'<br />
})<br />
// this ul will now be shrink-wrapped to longest li due to position:absolute<br />
// so save its width as ems. Clientwidth is 2 times faster than .width() - thanks Dan Switzer<br />
.end().end()[0].clientWidth / fontsize;<br />
// add more width to ensure lines don't turn over at certain sizes in various browsers<br />
emWidth += o.extraWidth;<br />
// restrict to at least minWidth and at most maxWidth<br />
if (emWidth > o.maxWidth) { emWidth = o.maxWidth; }<br />
else if (emWidth < o.minWidth) { emWidth = o.minWidth; }<br />
emWidth += 'em';<br />
// set ul to width in ems<br />
$ul.css('width',emWidth);<br />
// restore li floats to avoid IE bugs<br />
// set li width to full width of this ul<br />
// revert white-space to normal<br />
$LIs.css({<br />
'float' : liFloat,<br />
'width' : '100%',<br />
'white-space' : 'normal'<br />
})<br />
// update offset position of descendant ul to reflect new width of parent<br />
.each(function(){<br />
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<article><br />
<h1><p>Lab Protocol</p></h1><br />
</font><br />
<font face="Arial, Helvetica"><br />
<p><a href="#Site-Directed_Mutagenesis">1. Site-Directed Mutagenesis</a></p><br />
<p><a href="#Restriction">2. Mutation Verification by Restriction Enzyme Digestion</a></p><br />
<p><a href="#Media">3. Media Preparation</a></p><br />
<p><a href="#Bacterial">4. Bacterial Transformation</a></p><br />
<p><a href="#Colony">5. Colony PCR for Verification</a></p><br />
<p><a href="#Culture">6. Cultivate the Bacteria</a></p><br />
<p><a href="#Plasmid">7. Plasmid DNA Isolation</a></p><br />
<p><a href="#Restriction_2">8. Mutation Verification by Restriction Enzyme Digestion </a></p><br />
<p><a href="#Amplify">9. Polymerase Chain Reaction and Electrophoresis</a></p><br />
<p><a href="#Electrophoresis">10. Double Restriction Enzyme Digestion and Electrophoresis </a></p><br />
<p><a href="#Ligation">11. Ligation</a></p><br />
<p><a href="#Bacterial_Transformation">12. Bacterial Transformation</a></p><br />
<p><a href="#bacterial_colony">13. Bacterial Colony PCR</a></p><br />
<p><a href="#Culture_the">14. Cultivate the Bacteria</a></p><br />
<p><a href="#Plasmid_DNA">15. Plasmid DNA Isolation</a></p><br />
<p><a href="#Restriction_Enzyme">16. Restriction Enzyme Digestion and Electrophoresis</a></p><br />
<p><a href="#Ligation1">17. Ligation</a></p><br />
<p><a href="#Bacteria">18. Bacteria Transformation</a></p><br />
<p><a href="#Cultivate">19. Cultivate the Bacteria</a></p><br />
<p><a href="#Flow">20. Flow Cytometer Analysis</a></p><br />
<p><a href="#Fluorescence">21. Fluorescence Microscope </a></p><br />
<br/><br />
<h1></h1><br />
<br />
<p><br />
<a name="Site-Directed_Mutagenesis" ></a><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Site-Directed Mutagenesis</font></b></font></h3><br />
<br />
Plasmid pSB1A3 was chosen as a backbone vector for cloning. The Pst I site in pSB1A3 was mutated to Afl II site to facilitate following cloning processes. Proper primers were designed and PCR-based site-directed mutageneis were carried out to generate this mutation as descbibed below. <br/><br />
<br />
<!--<p>要到很后面才能找到他的结束--><br />
<strong>Method:</strong><br/><br />
<br />
1. Set up PCR assay tubes as described below:<br />
<br/><br />
Total: 25 μl<br /><br />
+ 0.25 μl of Ex Taq polymerase <br /><br />
+ 2.5 μl of 10× Taq reaction buffer<br /><br />
+ 2.0 μl of dNTP(2 mM) <br /><br />
+ 1.0 μl of template (E.coli plasmid 817) <br /><br />
+ 1.0 μl of oligonucleotide primer PtoA-F*<br /><br />
+ 1.0 μl of oligonucleotide primer PtoA-R* <br /><br />
+ 18.25 μl of ddH2O <br /><br />
*The sequences of primer pair PtoA-F and PtoA-R.<br /><br />
PtoA-F 5'-CCACCTGACGTCTAAGAAAC-3'<br /><br />
PtoA-R 5'-ATGATCATCGCCGGCGAATTCAGGC-3' <br /><br />
<br />
2. Set parameters for PCR to amplify desired products. <br/><br />
<br />
<table><br />
<tr><br />
<td>Temperature</td> <td>Time</td> <td>Cycle</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>5min</td> <td>1</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>55˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>72˚C</td> <td>1min 20sec</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>4˚C</td> <td>7hrs</td> <td>1</td><br />
</tr><br />
</table><br />
<!--那个p的结束,下同--><br />
</p><br />
<br/><br />
<br />
<p><br />
<font face="Arial, Helvetica"><br />
<br />
<a name="Restriction" ></a> <br />
</font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Restriction Enzyme Digestion for Verification</font></b></font></h3><br />
To verify whether PstI site in pSB1A3 was successfully mutated to AflII site, we performed restriction enzyme digestion experiments. <br/><br />
<br />
Bacterial plasmids are double-stranded circular DNA molecules and uncut plasmid DNA can be in any of three forms - nicked circular, linear, closed supercoiled. When run on an agarose gel one frequently will see these forms as different bands with closed supercoiled form migrates the fastest, linear form migrates the slowest, and nicked circular migrates in between. If the PStI site was successfully mutated to AflII site, we expected to see increased linear form of pSB1A3 when digested with AflII. SpeI-cut pSB1A3 was served as a positive control. <br /><br />
<br />
<strong>Method</strong><br /><br />
1. Set up Pst I digestion in 1.5 ml eppendorf tubes as described below:<br /><br />
Total: 10μl<br /><br />
+ 0.5μl of Pst I restriction enzyme (company :Takara)<br /><br />
+ 1μl of 10XH buffer<br /><br />
+ 1μl of plasmid DNA<br /><br />
+ 7.5μl of ddH2O <br /><br />
2. Set up Afl II digestion in 1.5 ml eppendorf tubes as described below:<br /><br />
Total: 10μl<br /><br />
+ 0.5μl of Afl II restriction enzyme, (company :Takara)<br /><br />
+ 1μl of 10XM buffer<br /><br />
+ 1μl of plasmid DNA<br /><br />
+ 1.0μl of 0.01% BSA<br /><br />
+ 6.5μl of ddH2O<br /><br />
3. Incubate the eppendorf tubes in 37℃ water bath for 1-2 hr. <br/><br />
4. Prepare 1% agarose gel in Conical flask. Weigh 0.6 g agarose and add 60 ml 1x TAE (diluted from 50x TAE). Cover the Conical flask with silver paper to avoid the loss of water vapor. Place the Conical flask in the microwave and microwave for 1 minute with a middle power. Take it out and shake gently till the solution is homogeneous,(BE CAREFUL to watch the solution closely when shake it–it superheats and can boil over and cause severe burns). Continue microwave and swirl until solution is seen clear and homogeneous with no existence of solid. After cool down the agarose gel briefly, add 3 μl of Gelred (10000x ) and mix well. Pour the agarose gel in gel casting apparatus and insert combs.<br/><br />
5. By inserting the pipette tip below the TAE liquid and into the well, add 5 μl of 1kb DNA ladder solution to first (and last if desired) well, skip one well, then begin adding the 5μl of digested DNA solutions mixed with 1 μl loading buffer (6x) to the wells. <br/><br />
6. Place the cover on the electrophoresis unit, plug into the power source, and turn on voltage to 120V, set time to 30 minutes, and press the start button twice,until the bubbles are seen. DNA separation can be observed as time goes on by turning off the power supply then gently removing the basin from the electrophoresis unit (be careful not to let the gel slip out of the basin) and placing on the UV transilluminator to see DNA bands. <br/><br />
7. When the desired level of separation is obtained, the basin can be placed on the transilluminator for picture taking(Of the absence of transilluminator,we use camera to take pictures with the UV light ). <br/><br />
8. Cut the gel of specific position and collect it in tubes that have measured weight. <br/><br />
9. Use DNA Gel Extraction Ki to purify the plasmid DNA mutant-pSB1A3.<br />
RPF (SpeI/AflII-digested), GFP (SpeI/AflII-digested) fragments were ligated into this vector, which was followed by insertion of designed terminator sequences between RFP and GFP, respectively.</p><br />
<font face="Arial, Helvetica"><br />
<img src="https://static.igem.org/mediawiki/2012/5/59/11.png" alt="" class="img_fl img_border" align="left"/> <br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
(Figure 1 : This figure shows that the site-directed mutagenesis succeed ,we successfully change a restriction enzyme cutting site named Pst I to Afl II. Lane 1 represents the plasmid mutant-pSB1A3, lane 2 shows that the mutant-pSB1A3 cannot be digested by restriction enzyme Spe I ,lane 3 shows that mutant-pSB1A3 can be digested by restriction enzyme Afl II.) <br />
</p><br />
<br/><br />
<br />
<p><br />
<a name="Media"></a><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Media Preparation</font></b></font></h3><br />
For all experiments involving the bacterial biomass and experimentation, proper media is chosen to grow the cells. Commonly,we use Lysogeny broth media for <em>E. coli</em>. The following is the media compositions and their quantities.<br /><br />
<strong>Method:</strong><br/><br />
<br />
1.Prepare the Lysogeny Broth (LB) liquid media (1 L) as indicated below: <br/><br />
+ Bacto-Tryptone - 10 g<br/><br />
+ NaCl - 10 g<br/><br />
+ Yeast Extract - 5 g<br/><br />
Add ddH2O and 5mmol/L Tris Buffer in a measuring cylinder to ensure accuracy, to make a total of 1 liter and pH is 8.0.<br/><br />
2.Prepare the Lysogeny Broth (LB) solid media (1 L) as indicated below:<br/><br />
+ Bacto-Tryptone - 10 g<br/><br />
+ NaCl - 10 g<br/><br />
+ Yeast Extract - 5 g<br/><br />
+ Difco Agar - 15g<br/><br />
Add ddH2O and 5mmol/L Tris Buffer in a measuring cylinder to ensure accuracy, to make a total of 1 liter and pH is 8.0.<br/><br />
3. Autoclaving<br/><br />
Autoclave at 121 °C for 60 minutes. After the media cooling down enough, antibiotics Ampicillin(100mg of Ampicillin per 1ml of the media) are added. At last the media are poured 15ml on each plate and become solid.Store the plate at 4℃ refrigerator.<br />
</p><br />
<br/><br />
<br />
<p><br />
<font face="Arial, Helvetica"><br />
<a name="Bacterial"></a></font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Bacterial Transformation</font></b></font></h3><br />
<br />
Transformation is commonly used to introduce recombinant plasmid DNA into bacterial strains which can transform naturally or can be made competitive for transformation by artificial means. The purpose of this technique is to introduce a recombinant plasmid DNA into a bacterial strains and to use bacteria strains to amplify the plasmid mutant-pSB1A3 for further plasmid construction.<br />
<br />
<strong>Method</strong><br /><br />
1. Take out an appropriate number of tubes that contain competent cells(100μl ) from the freezer. Immediately place the tubes on ice, so that all but the cap is surrounded by ice. Allow the cells to thaw on ice for 2-5 min.<br /><br />
2. Visually check the cells to see whether they have thawed and gently flick the cells 1-2 times to evenly resuspend the cells.<br /><br />
3. Add 10μl PCR products(mini-prep purified) to the competent cells DH-5α. Stir gently to mix and return the tube to the ice, making sure that the tube is surrounded by ice except for the cap. Repeat for additional two times for the same samples.<br /><br />
4. Incubate the tubes on ice for 30 min.<br /><br />
5. Place the tubes in a 42°C water bath for exactly 90 sec; do not shake.<br /><br />
6. Place the tubes on ice for 2 min to cool down.<br /><br />
7. Add 800 μl of room temperature LB medium to each tube.<br /><br />
8. Shake the tubes vigorously at 37°C for 45-60 min.<br /><br />
9. Centrifuge the tubes at 3K RPM for 1 min. Discard the supernatant liquor and leave 100-200 μl of the mixtures.Mix the contents and spread the whole liquid on LB agar plates containing the appropriate antibiotic ampicillin for the plasmid.<br /><br />
10. Place the plates on the bench for several min to allow excess liquid to be absorbed, and then invert and incubate overnight at 37°C (12-16 h).<br />
<br/><br />
</p><br />
<br />
<p><br />
<a name="Colony"></a><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Colony PCR for Verification</font> </b></font></h3><br />
<br />
Colony PCR is used to identify and select cell colonies that have the correct plasmid inserted. The procedure is a way to do several PCR operations on cell colonies in parallel, to evaluate the results and select the corresponding positive cell colonies. After an overnight growth of E.coli, we can pick up several colonies from the plate and do a colony PCR verification. Besides, the colonies we choose and should also be stored, we can incubate these colonies in one plate after every colony has been marked.<br/><br />
<br />
<strong>Method</strong><br /><br />
1. Prepare the sample reaction as indicated below:<br /><br />
Total: 25μl <br /><br />
+ 0.25 μl of Ex Taq polymerase (company:Takara)<br /><br />
+ 2.5 μl of 10× Taq reaction buffer<br /><br />
+ 1.0 μl of R-NPS-F*<br /><br />
+ 1.0 μl of G-SXA-R*<br /><br />
+ 1.0 μl of plasmid mutant-pSB1A3<br /><br />
+ 2.0 μl of dNTP( 25mM ) <br /><br />
+ 17.25 μl of ddH2O <br /><br />
Note: The sequences of primers R-NPS-F , G-SXA-R <br /><br />
R-NPS-F 5'-TATAGCGGCCGCCTTAAGTAAGTAAGAGTATACG-3' <br /><br />
G-SXA-R 5'-TCTCCGACTTAAGGGATCCTATAAACGCAG-3'<br /><br />
<br />
2. Set parameters for PCR to amplify desired products. <br />
<br />
<table><br />
<tr><br />
<td>Temperature</td> <td>Time</td> <td>Cycle</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>5min</td> <td>1</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>55˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>72˚C</td> <td>1min 20sec</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>4˚C</td> <td>7hrs</td> <td>1</td><br />
</tr><br />
</table><br />
<br />
3. Electrophorese the total system and observe the lane separation. <br />
</p><br />
<br/><br />
<br />
<p><br />
<a name="Culture"></a><br />
<br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Cultivate the Bacteria</b></font></h3><br />
According to the results of the PCR detection, positive colonies are chosen and transferred them to 5ml LB liquid media ( 5μl of ampicillin added) stored in 12.5ml centrifuge tubes. Put the centrifuge tubes in 37℃ gas bath overnight. <br />
</p><br />
<br/><br />
<br />
<p><br />
<a name="Plasmid"></a><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Plasmid DNA Isolation</b></font></h3><br />
Use E.Z.N.A.TM Plasmid Mini I to realize plasmid DNA isolation.<br /><br />
<strong>Method</strong><br/><br />
<br />
<ol><br />
<li>Transfer 5 ml of overnight culture into a 1.5-ml eppendorf tube labeled with group number. </li><br />
<li>Centrifuge the sample at max. speed of desk top centrifuge and RT for 1min to pellet the cells.</li><br />
<li>Discard the supernatant. Remove as much of the supernatant as possible without disturbing the cell pellet. </li><br />
<li>Repeat step 1 and 2 twice. </li><br />
<li>Resuspend the pellet completely in 250 ml of Solution I (containing RNase A) by vortexing the samples vigorously . No clumps should be visible in the tube. </li><br />
<li>Add 250 ml of Solution II and mix the sample by gently inverting the tube 4 to 6 times. Do not vortex or shake the sample vigorously. The bacterial suspension should begin to clear which have lysed the bacterial cells in this step. <strong>Warning: </strong>Do not stop here for more than five min, as the high pH hurts your DNA! </li><br />
<li>Add 350 ml of Solution III and mix by gently inverting the tube 4 to 6 times until a flocculent white precipitate forms. Do not shake vigorously, as it might break the genomic DNA. </li><br />
<li>Centrifuge at maximum speed for 10 min at room temperature to pellet the cell debris. You should see a white precipitate in the tube after the centrifugation. </li><br />
<li>While the samples are centrifuging, for each sample, label a clean HiBind Miniprep Column which is to assembled in a 2-ml collection tube</li><br />
<li>Apply the supernatants from step 8 to the columns. </li><br />
<li>Centrifuge at maximum speed for 1 min at RT. Discard the flow-through in the collection tube. </li><br />
<li>Add 500 ml of Buffer HB to wash the Hibind Miniprep Column. Centrifuge at maximum speed for 1 min at RT. Discard the flow-through in the collection tube. </li><br />
<li>Wash the column by adding 700 ml of DNA Wash Buffer diluted with absolute ethanol. Centrifuge at maximum speed for 1 min at room temperature and discard the flow-through.</li><br />
<li>Then centrifuge the tubes again for 2 min to remove all the moisture.</li><br />
<li>Place the column in a clean 1.5 ml eppendorf tube that is labeled with the plasmid name and group number. To elute the DNA, add 50 ml of Elution Buffer to the center of each column. Let the samples stand for 2 or more minutes at RT, and then centrifuge for 1 min. The sample in the centrifuge tube (bottom) is your plasmid DNA. </li><br />
<li>Discard the column and save the sample in the eppendorf tube by placing it in the freezer (-20°C). </li><br />
</ol><br />
</p><br />
<br />
<p><br />
<a name="#Restriction_2" ></a> <br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Restriction Enzyme Digestion and Electrophoresis</font></b></font></h3><br />
<br />
Because the colony PCR test is so sensitive and affect markedly by environment factors. So we do a restriction enzyme digestion to ensure that the isolated plasmid is the site-directed mutated plasmid.<br /><br />
<strong>Method</strong><br /><br />
1. Prepare the control reaction as indicated below:<br /><br />
Total: 10μl<br /><br />
+ 0.5μl of Pst I restriction enzyme(company :Takara)<br /><br />
+ 1μl of 10XH buffer<br /><br />
+ 1μl of plasmid DNA<br /><br />
+ 7.5μl of ddH2O <br /><br />
2. Prepare the sample reaction as indicated below:<br /><br />
Total: 10μl<br /><br />
+ 0.5μl of Afl II restriction enzyme, (company :Takara)<br /><br />
+ 1μl of 10XM buffer<br /><br />
+ 1μl of plasmid DNA<br /><br />
+ 1.0μl of 0.01% BSA<br /><br />
+ 6.5μl of ddH2O <br /><br />
3. Electrophorese the total system and observe the lane separation.<br />
</p><br />
<br/><br />
<br />
<p><br />
<a name="Amplify"></a> <br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Polymerase Chain Reaction(GFP &amp; RFP) and Electrophoresis</font></b></font></h3><br />
<br />
GFP and RFP DNA fragments are the insert which need to be ligate to the plasmid mutant-pSB1A3. Do a PCR amplification can get enough quantities for the following reactions.<br /><br />
<strong>Method </strong><br /><br />
1.Prepare the sample reaction as indicated below:<br /><br />
Total: 100μl ( PCR amplification of GFP fragments) <br /><br />
+ 1.0μl of Taq DNA polymerase,#EP0402<br /><br />
+ 10μl of 10XTaq buffer <br /><br />
+ 10μl of MgCl2(25mM)<br /><br />
+ 10μl of dNTP(2mM)<br /><br />
+ 2μl of G-SXA-R* <br /><br />
+ 2μl of G-SXA-F*<br /><br />
+ 4μl of DNA template<br /><br />
+ 61μl of ddH2O <br /><br />
Total: 100μl(PCR amplification of RFP fragments) <br /><br />
+ 1.0μl of Taq DNA polymerase, EP0402<br /><br />
+ 10μl of 10XTaq buffer <br /><br />
+ 10μl of MgCl2(25mM)<br /><br />
+ 10μl of dNTP(2mM)<br /><br />
+ 2μl of R-NPS-R* <br /><br />
+ 2μl of R-NPS-F*<br /><br />
+ 4μl of DNA template<br /><br />
+ 61μl of ddH2O <br /><br />
<br />
Note: The sequences of primers R-NPS-F , R-NPS-R , G-SXA-F ,G-SXA-R <br /><br />
R-NPS-F 5'-TATAGCGGCCGCCTTAAGTAAGTAAGAGTATACG-3' <br /><br />
R-NPS-R 5'-CGGAGACTAGTCTGCAGATCACATAAGTAAAGTGATAATC-3' <br /><br />
G-SXA-F 5'-CTAGACTAGTTCTAGAGGCGGACTCACTATAGA-3' <br /><br />
G-SXA-R 5'-TCTCCGACTTAAGGGATCCTATAAACGCAG-3'<br /><br />
<br />
2. Set parameters for PCR to amplify desired products. <br/><br />
<br />
<table><br />
<tr><br />
<td>Temperature</td> <td>Time</td> <td>Cycle</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>5min</td> <td>1</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>55˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>72˚C</td> <td>1min 20sec</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>4˚C</td> <td>7hrs</td> <td>1</td><br />
</tr><br />
</table><br />
<br />
<br />
3. Use DNA Gel Extraction Kit to purify the GFP and RFP DNA fragments after the Electrophoresis.<br />
<br />
<font face="Arial, Helvetica"><img src="https://static.igem.org/mediawiki/2012/7/72/111.png" alt="" class="img_fl img_border" align="left"/> </font><br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
(Figure 2 : This figure shows that The PCR reaction system can amplify large quantities of GFP and RFP DNA fragments. The digestion based on the GFP and RFP DNA fragments can be done to prepare for the ligation. Lane 1 represents the template E.coli 817 can amplify the GFP and RFP DNA fragments , lane 2 represents the template E.coli 817(355.5) can also amplify the GFP and RFP DNA fragments.)<br/><br/><br />
</p><br />
<br/><br />
<br />
<p><br />
<font face="Arial, Helvetica"><br/><br />
<a name="Restriction_Enzyme"></a><br />
</font><br />
<br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Double Restriction Enzyme Digestion and Electrophoresis.</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
Use specific restriction enzymes to digest plasmid mutant-pSB1A3,GFP and RFP to get sticky ends and purify the DNA fragment after the Electrophoresis.<br /><br />
<strong>Method:</strong><br/><br />
</font><br />
<br />
1. Digestion of plasmid mutant-pSB1A3 <br/><br />
Prepare the sample reaction as indicated below:<br /><br />
Total: 50μl <br /><br />
+ 3.0μl of Not I restriction enzyme, #ER0591<br /><br />
+ 3.0μl of Afl II restriction enzyme, #ER0831<br /><br />
+ 5.0μl of 10X buffer O <br /><br />
+ 1.0μl of mutant-pSB1A3 plasmid <br /><br />
+ 37.0μl of ddH2O<br /><br />
<br />
2. Digestion of PCR product GFP<br /><br />
Prepare the sample reaction as indicated below:<br /><br />
Total: 50μl <br /><br />
+ 3.0μl of Not I restriction enzyme, #ER0591<br /><br />
+ 3.0μl of Spe I restriction enzyme, #ER1251 <br /><br />
+ 5μl of 10X buffer Tango<br /><br />
+ 10μl of PCR products GFP<br /><br />
+ 29μl of ddH2O<br /><br />
<br />
3. Digestion of PCR product RFP<br /><br />
Prepare the sample reaction as indicated below:<br /><br />
Total: 50μl <br /><br />
+ 3.0μl of Afl II restriction enzyme, #ER0831<br /><br />
+ 3.0μl of Spe I restriction enzyme, #ER1251 <br /><br />
+ 5μl of 10X buffer Tango<br /><br />
+ 10μl of PCR products RFP<br /><br />
+ 29μl of ddH2O<br /><br />
<br />
4. Put the tubes in 37℃ environment for 4-8 hours <br /><br />
<br />
5. Use DNA Gel Extraction Kit to purify the mutant-pSB1A3 fragment, GFP and RFP after digestion and named them by mutant-pSB1A3 (NA) ,GFP(NS) and RFP(AS) after the Electrophoresis.<br />
</p><br />
<br/><br />
<br />
<p><br />
<font face="Arial, Helvetica"><br />
<a name="Ligayion"></a> </font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Ligation</font></b></font></h3><br />
<br />
Ligation is the process that target DNA gene is inserted into a plasmid. Both the vector and insert are prepared to have the sticky ends. These two kinds of DNA pieces are placed in a reaction tube and the proper DNA ligase, buffer, and cofactors are added for the reaction to take place. When done properly, the ligation will result in a successful combination of the insert and plasmid into one plasmid.Based on the digestion of mutant-pSB1A3,GFP and RFP DNA fragments,also ligation can be done. We ligate mutant-pSB1A3 vector and sticky GFP and RFP DNA fragments to construct an new plasmid mutant-pSB1A3-GR. <br /><br />
<br />
1. Prepare the control reaction as indicated below:<br /><br />
Total: 10μl <br /><br />
+ 1.0μl of plasmid mutant-pSB1A3 (NA) <br /><br />
+ 3.0μl of GFP(NS) <br /><br />
+ 3.0μl of RFP(AS) <br /><br />
+ 2.0μl of T4 DNA Ligase,#EL0011 <br /><br />
+ 1.0μl of 10XT4 Ligase buffer<br /><br />
Note: GFP(NS) means the product of GFP DNA fragments digested by restriction enzyme Not I and Spe I. <br /><br />
2. Prepare the sample reaction as indicated below:<br /><br />
Total: 10μl <br /><br />
+ 1.0μl of plasmid mutant-pSB1A3 (NA) <br /><br />
+ 2.0μl of T4 DNA Ligase, #EL0011 <br /><br />
+ 1.0μl of 10XT4 Ligase buffer<br /><br />
+ 6.0μl of ddH2O<br /><br />
3. Put the tubes in 22℃ water bath, react for 8-12 hours.<br />
</p> <br/><br />
<br />
<p><br />
<a name="Bacterial_Transformation"></a> <br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Bacterial Transformation</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
Transform the ligation products into the DH-5α competent cells, put the plate on 37℃ gas bath for 12-16 hours. <br />
</p><br />
<br/><br />
<br />
<p><br />
<a name="Bacterial_Colony"></a> </font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Bacterial Colony PCR</font></b> </font></h3><br />
<br />
Colony PCR is used to identify and select cell colonies that have the correct plasmid insert. This procedure is a way to do several PCR operations on cell colonies in parallel, to evaluate the results and select the corresponding good cell colonies. After an overnight growth of E.coli, we can pick up some colonies from the plate and do a colony PCR verification. Besides, the colonies we choose and should also be stored, we can incubate these colonies in one plate after every colony has been marked.<br /><br />
<strong>Method</strong><br /><br />
1. Prepare the sample reaction as indicated below:<br /><br />
Total: 20μl <br /><br />
+ 0.25 μl of Ex Taq polymerase,#EP0402 <br /><br />
+ 2.0 μl of 10× Taq reaction buffer<br /><br />
+ 1.0 μl of R-NPS-F* <br /><br />
+ 1.0 μl of G-SXA-R*<br /><br />
+ 5.0 μl of bacterial colony<br /><br />
+ 2.0 μl of dNTP( 25mM ) <br /><br />
+ 8.75 μl of ddH2O <br /><br />
Note: The sequences of primers R-NPS-F , R-NPS-R , G-SXA-F ,G-SXA-R <br /><br />
R-NPS-F 5'-TATAGCGGCCGCCTTAAGTAAGTAAGAGTATACG-3' <br /><br />
R-NPS-R 5'-CGGAGACTAGTCTGCAGATCACATAAGTAAAGTGATAATC-3' <br /><br />
G-SXA-F 5'-CTAGACTAGTTCTAGAGGCGGACTCACTATAGA-3' <br /><br />
G-SXA-R 5'-TCTCCGACTTAAGGGATCCTATAAACGCAG-3'<br /><br />
<br />
2. Set parameters for PCR to amplify desired products. <br/> <br />
<table><br />
<tr><br />
<td>Temperature</td> <td>Time</td> <td>Cycle</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>13.5min</td> <td>1</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>55˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>72˚C</td> <td>1min 20sec</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>4˚C</td> <td>7hrs</td> <td>1</td><br />
</tr><br />
</table><br />
<br />
3. Electrophorese the total system and observe the lane separation. <br/><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/0/07/1111.png" alt="" class="img_fl img_border" align="left"/> <br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
(Figure 3: The lane on the figure are 2k DNA fragments, it shows the GFP and RFP DNA fragments are ligated to the vectors which were isolated from the bacterial colonies.) <br />
</p><br />
<br/><br />
<br />
<p><br />
<font face="Arial, Helvetica"><br />
<a name="Culture_the"></a><br />
</font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Cultivate the Bacteria</b></font></h3><br />
According to the results of the PCR detection, we choose positive colonies and transfer them to 5ml LB liquid media ( 5μl of ampicillin has added) stored in 12.5ml centrifuge tubes. Put the centrifuge tubes in 37℃ gas bath overnight.<br />
</p><br />
<br/><br />
<br />
<p><br />
<font face="Arial, Helvetica"><a name="Plasmid_DNA"></a> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Plasmid DNA Isolation</b></font></h3><br />
<font face="Arial, Helvetica">Use E.Z.N.A.TM Plasmid Mini I to isolate the constructed plasmid mutant-pSB1A3-GR. </font><br />
</p><br />
<br/><br />
<br />
<p><br />
<font face="Arial, Helvetica"><a name="Restriction_Enzyme" ></a> </font><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Restriction Enzyme Digestion and Electrophoresis</b></font></h3><br />
From the last step, we got the certain quantities of isolated plasmids. In this step, we do two restriction enzyme digestion reactions, one to prove that the plasmid is construct correctly ( mutant-pSB1A3-GR ), one to get sticky ends preparing for the ligation.<br /><br />
<strong>Method</strong><br />
1.Restriction Enzyme Digestion to prove that plasmid is constructed correctly<br />
<br />
1.1. Prepare the sample reaction as indicated below:<br /><br />
Total: 10μl<br /><br />
+ 1.0μl of Not I restriction enzyme,#ER0591<br /><br />
+ 1.0μl of Spe I restriction enzyme,#ER1251 <br /><br />
+ 2.0μl of Buffer Tango( 10X )<br /><br />
+ 1.5μl of plasmid mutant-pSB1A3-GR<br /><br />
+ 14.5μl of ddH2O <br /><br />
1.2. Electrophorese the total system and observe the lane separation. <br/><br />
<br />
2. Restriction Enzyme Digestion to get sticky ends preparing for the ligation.<br/><br />
<br />
2.1. Prepare the sample reaction as indicated below:<br /><br />
Total: 50μl<br /><br />
+ 5.0μl of Pst I restriction enzyme, #ER0611<br /><br />
+ 5.0μl of Xba I restriction enzyme, #ER0681 <br /><br />
+ 3.0μl of Buffer Tango( 10X )<br /><br />
+ 5.0μl of plasmid mutant-pSB1A3-GR<br /><br />
+ 32.0μl of ddH2O <br /><br />
2.2. Electrophorese the total system and observe the lane separation.<br /><br />
2.3. Cut the gel of specific position and collect it in tubes that have measured weight. <br /><br />
2.4. Use DNA Gel Extraction Ki to purify the plasmid DNA mutant-pSB1A3-GR(PX) .<br /><br />
Note: Mutant-pSB1A3-GR(PX) means plasmid Mutant-pSB1A3-GR digested by Pst I and Xba I.<br />
<br />
<br />
<strong><img src="https://static.igem.org/mediawiki/2012/d/d8/11111.png" alt="" class="img_fl img_border" align="left" /></strong><br />
(Figure 4 : The double digestion of mutant-pSB1A3 forms a linear DNA fragments and it runs slower than circle DNA fragments. This suggest that the double digestion of mutant-pSB1A3 works in a high efficiency, and desired sticky ends are formed.1,3,5 are plasmids digested by restriction enzyme Pst I and Xba I from different colonies, 2,5,6 are pure plasmid mutant-pSB1A3-GR, 7 is the plasmid mutant-pSB1A3. )<br />
</p><br />
<br/><br />
<br />
<p><br />
<font face="Arial, Helvetica"><a name="Ligation1" ></a> </font><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Ligation</b></font></h3><br />
Ligation is the process that target DNA gene is inserted into a plasmid. Both the vector and insert are prepared to have the sticky ends. These two kinds of DNA pieces are placed in a reaction tube and the proper DNA ligase, buffer, and cofactors are added for the reaction to take place. When done properly, the ligation will result in a successful combination of the insert and plasmid into one plasmid.Based on the digestion of mutant-pSB1A3-GR, terminator DNA fragments,ligation can be done. We ligate mutant-pSB1A3-GR vector and sticky terminator DNA fragments to construct an new plasmid mutant-pSB1A3-GR-t.By detecting the quantities of GFP and RFP, terminator efficiency can be calculated. <br /><br />
1. Prepare the control reaction as indicated below:<br /><br />
Total: 10μl <br /><br />
+ 1.0μl of plasmid mutant-pSB1A3 (NA) <br /><br />
+ 6.0μl of terminator<br /><br />
+ 2.0μl of T4 DNA Ligase , #EL0011 <br /><br />
+ 1.0μl of 10XT4 Ligase buffer<br /><br />
2. Put the tubes in 22℃ water bath, react for 8-12 hours. <br />
</p><br />
<br/><br />
<br />
<p> <br />
<font face="Arial, Helvetica"><a name="Bacteria" id="Culture_the"></a> </font><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Bacteria transformation</b></font></h3><br />
Transform the ligation products into the DH-5α competent cells, put the plate on 37℃ gas bath for 12-16 hours. <br />
</p><br/><br />
<br />
<p><br />
<font face="Arial, Helvetica"><a name="Cultivate"></a> </font><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Cultivate the Bacteria</b></font></h3><br />
According to the results of the PCR detection, we choose positive colonies and transfer them to 5ml LB liquid media ( 5μl of ampicillin has added) stored in 12.5ml centrifuge tubes. Put the centrifuge tubes in 37℃ gas bath overnight.<br />
</p> <br/><br />
<br />
<p><br />
<font face="Arial, Helvetica"><a name="Flow" ></a> </font><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Flow Cytometer Analysis</b></font></h3><br />
Flow cytometry is a laser based, biophysical technology employed in cell counting, sorting, biomarker detection and protein engineering, by suspending cells in a stream of fluid and passing them by an electronic detection apparatus. It allows simultaneous multiparametric analysis of the physical and/or chemical characteristics of up to thousands of particles per second. <br/><br />
Each fluorophore has a characteristic peak excitation and emission wavelength, and the emission spectra often overlap. Consequently, the combination of labels which can be used depends on the wavelength of the lamp(s) or laser(s) used to excite the fluorochromes and on the detectors available. It is practical that the expression of RFP and GFP can be measured at the same time.<br/><br />
<strong>Method:</strong><br/><br />
1. Materials:<br/><br />
&nbsp;&nbsp;1.1. NaCl solution( 0.9% ,M/V)<br/><br />
&nbsp;&nbsp;1.2. 75% ethanol<br/><br />
2. Procedures:<br/><br />
&nbsp;&nbsp;2.1. Bacterium are harvested by centrifuge at 10000g for 30s, then discard the supernatant. Repeat this step again until we got adequate bacterium.<br/><br />
&nbsp;&nbsp;2.2. Suspend the bacterium With NaCl solution(0.9%) and vibrate the centrifuge tubes until the bacterium are distributed homogeneous.<br/><br />
&nbsp;&nbsp;2.3. Load the bacteria containing pSB1A3 to Flow Cytometer (Beckman), set the parameters. Measure the GFP-FL1 and RFP-FL2 by Cytometer. <br />
</p><br />
<br/><br />
<br />
<p><br />
<font face="Arial, Helvetica"><a name="Fluorescence"></a> </font><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Fluorescence Microscope</b></font></h3><br />
A fluorescence microscope is an optical microscope that uses fluorescence and phosphorescence reflection and absorption to study properties of organic or inorganic substances and generate an image.Fluorescence microscope detection are used to confirm the expression of GFP and RFP which are illuminated with light of a wavelength which excites fluorescence in the sample.<br />
<br/><br />
<strong>Method</strong><br /><br />
Add 10μl of bacteria solution to micro slide and cover with coverslip.<br />
Placed the micro slide on the Fluorescence Microscope. Set parameters to detect GFP and RFP. <br/><br />
Generate an image of fluorescence protein and save it.<br />
</p><br />
<br/> <br />
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</html></div>M.B.ZHOUhttp://2012.igem.org/Team:SUSTC-Shenzhen-B/protocol1Team:SUSTC-Shenzhen-B/protocol12012-10-26T21:43:50Z<p>M.B.ZHOU: </p>
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#menubar {display:none; }<br />
<br />
/* googleapis-font */<br />
@font-face {<br />
font-family: 'Open Sans';<br />
font-style: normal;<br />
font-weight: 700;<br />
src: local('Open Sans Bold'), local('OpenSans-Bold'), url(http://themes.googleusercontent.com/static/fonts/opensans/v6/k3k702ZOKiLJc3WVjuplzHhCUOGz7vYGh680lGh-uXM.woff) format('woff');<br />
}<br />
@font-face {<br />
font-family: 'Open Sans';<br />
font-style: normal;<br />
font-weight: 600;<br />
src: local('Open Sans Semibold'), local('OpenSans-Semibold'), url(http://themes.googleusercontent.com/static/fonts/opensans/v6/MTP_ySUJH_bn48VBG8sNSnhCUOGz7vYGh680lGh-uXM.woff) format('woff');<br />
}<br />
@font-face {<br />
font-family: 'Open Sans';<br />
font-style: normal;<br />
font-weight: 400;<br />
src: local('Open Sans'), local('OpenSans'), url(http://themes.googleusercontent.com/static/fonts/opensans/v6/cJZKeOuBrn4kERxqtaUH3T8E0i7KZn-EPnyo3HZu7kw.woff) format('woff');<br />
}<br />
<br />
/* inner */<br />
.pagenavi{clear:both;padding:0}.pagenavi a,.pagenavi a:visited{background:#dfddc7;display:inline-block;margin:0 8px 0 0;padding:3px 10px;color:#727272}.pagenavi a:hover{background:#dfddc7;display:inline-block;margin:0 8px 0 0;padding:3px 10px;color:#b03121;text-decoration:none}.pagenavi .current{background:#dfddc7;display:inline-block;margin:0 8px 0 0;padding:3px 10px;color:#b03121}.pagenavi .pages{background:#dfddc7;display:inline-block;margin:0 8px 0 0;padding:3px 10px}#breadcrumb{text-align:right;margin-bottom:34px;clear:right}.post{margin-bottom:30px;padding-bottom:30px;clear:both;border-bottom:1px solid #dfddc7}.post img{padding:5px;background:#d2d0ba;width:590px;height:236px}.single{padding-bottom:20px}.postimg{margin-bottom:20px}.posttitle{margin:0 0 5px 0}.posttitle,.posttitle a{font-size:18px;color:#3f4432}.posttitle a:hover{text-decoration:none}.entry-text{overflow:hidden;padding-left:30px}.entry-text h2{padding-top:5px}.entry-content{margin:0;padding:12px 0 5px 0}.entry-date{float:left;text-align:center;font-size:18px;width:56px;height:48px;padding-top:8px;line-height:normal;-moz-border-radius:56px;-webkit-border-radius:56px;-khtml-border-radius:56px;border-radius:56px;background:#3f4432;color:#f1efda;display:block}.month{font-size:11px;display:block}.entry-utility{padding:20px 0 0;font-size:11px;font-style:italic}.single .entry-utility{padding:0 0 0}#comment h2{margin-bottom:25px;text-transform:uppercase;font-size:14px}.commentlist{list-style-type:none;padding:0;margin:0}.commentlist ol{list-style-type:none;padding:30px 0 0 90px;margin:0}.commentlist li{position:relative;padding:0 0 30px 0}.commentlist li li{position:relative;padding:0}.avatar{position:absolute;top:0;left:0}.fn{font-size:12px;font-style:normal}.tdate{padding-left:30px}.tdate,.reply{font-size:11px;color:#a6a6a6;font-style:italic}.comment-body{margin:0 0 0 90px;padding:20px;background:#f7f5e3}.comment-body p{margin-bottom:5px;margin-top:10px}.comment-body .more{padding:0 0}#commentform{margin-bottom:15px}#commentform label{display:block}#commentform .text-input{margin-bottom:8px;padding:8px 5px;vertical-align:middle}#commentform .textarea{margin-bottom:20px;padding:8px 5px;vertical-align:top;width:90%}.ts-gallery-img img{max-width:100%;padding:0;margin:0 auto}.ts-gallery-clear{clear:both;height:1px!important;line-height:1px!important;float:none!important}.ts-gallery ul{list-style-type:none;margin:0;padding:0;clear:both}.ts-gallery ul li.nomargin{margin-right:0}.ts-gallery-text{padding:3px 0;text-align:center}.ts-gallery-text h2{font-size:13px;color:#3f4432;margin-bottom:20px;font-weight:600;font-family:'Open Sans',sans-serif}.no-gallery-text{display:none}.ts-gallery-img{background:#d2d0ba;padding:5px;position:relative;line-height:normal}.ts-gallery-img:hover{background:#3f4432}.ts-gallery-img a.image{display:block;position:relative;overflow:hidden;line-height:normal}.ts-gallery-img a .rollover{background:url(/wiki/images/5/5d/Sustc-b1-hover-zoom.png);background-color:#000;background-repeat:no-repeat;background-position:center;display:block;position:absolute;z-index:10;display:none;cursor:pointer}.ts-gallery-img a .rollover.gotopost{background:url(/wiki/images/e/ec/Sustc-b1-hover-doc.png);background-color:#000;background-repeat:no-repeat;background-position:center}.ts-gallery-col4{list-style-type:none;padding:0;margin:0}.ts-gallery-col4 li{list-style-type:none;padding:0;margin:0 20px 0 0;width:220px;float:left}.ts-gallery-col4 li.nomargin{margin-right:0}.ts-gallery-col4 .ts-gallery-img{width:210px;height:81px}.ts-gallery-col4 .ts-gallery-img img{width:210px;height:81px}.ts-gallery-col4 .ts-gallery-img a.image{width:210px;height:81px;display:block;position:relative}.ts-gallery-col4 .ts-gallery-img a .rollover{width:210px;height:81px}.ts-gallery-col3{list-style-type:none;padding:0;margin:0}.ts-gallery-col3 li{list-style-type:none;padding:0;margin:0 20px 25px 0;width:300px;float:left}.ts-gallery-col3 li.nomargin{margin-right:0}.ts-gallery-col3 h2{margin:0 0 5px 0!important}.ts-gallery-col3 .ts-gallery-img{width:290px;height:115px}.ts-gallery-col3 .ts-gallery-img img{width:290px;height:115px}.ts-gallery-col3 .ts-gallery-img a.image{width:290px;height:115px;display:block;position:relative}.ts-gallery-col3 .ts-gallery-img a .rollover{width:290px;height:115px}.ts-gallery-col3 .ts-gallery-text{padding:5px 0 0 0}form{margin:0;padding:0}fieldset{border:0}#contactform{margin:0 auto;position:relative}#contactform h4{text-transform:uppercase;margin-bottom:20px}#contactform label{display:block;width:100%;float:left;padding-bottom:5px}span.required{color:#888}span.error{color:red;text-align:left;font-size:11px;padding-bottom:15px;display:block}#contactform input.text-input{margin-bottom:15px;vertical-align:middle;width:60%;float:left;font-style:italic;padding:8px;color:#727272;font-size:11px}#contactform textarea{width:95%;float:left;font-style:italic;color:#727272;font-size:11px;font-family:Arial,Helvetica,sans-serif}#message{margin-left:0;font-weight:700;color:red}#message h2{}#message p{margin:6px 0}.note{color:#d45454}#contactform .button{cursor:pointer;margin-top:20px;clear:both;float:left}<br />
<br />
/* style */<br />
html,body{height:100%}body{font-family:Arial,Helvetica,sans-serif;font-size:12px;color:#727272;margin:0;padding:0;line-height:20px;background:#d6d4c0 url(/wiki/images/d/d7/Sustc-b1-pattern.png) repeat}*{margin:0;padding:0}:focus{outline:0}form{margin:0;padding:0}hr{border-width:0;height:1px;line-height:0;margin:30px 0;page-break-after:always;text-align:center;width:100%;clear:both;color:#dfddc7;background-color:#dfddc7}.clearfix:before,.clearfix:after{content:'\0020';display:block;overflow:hidden;visibility:hidden;width:0;height:0}.clearfix:after{clear:both}.clearfix{zoom:1}.clear,.clr{clear:both;display:block;overflow:hidden;visibility:hidden;width:0;height:0}h1,h2{margin-bottom:25px}h3,h4,h5,h6{margin-bottom:10px}h1{font-size:26px}h2{font-size:20px}h3{font-size:16px}h4{font-size:14px}h5{font-size:12px}h6{font-size:10px}h1,h2{font-weight:400;font-family:'Open Sans',sans-serif;color:#404631}h3,h4,h5,h6{font-weight:600;font-family:'Open 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0;padding:0 10px 0 50px;background-image:url(/wiki/images/3/3b/Sustc-b1-quote.png);background-repeat:no-repeat;background-position:0 0;clear:both;font-family:Georgia,Arial;font-style:italic;font-size:16px;line-height:22px}blockquote.left,blockquote.right{float:right;letter-spacing:0;margin-bottom:20px;margin-left:20px;margin-top:0;padding:0 20px 10px 60px;width:43%;background-position:0 0}blockquote.left{float:left;margin-left:0;margin-right:20px}blockquote p{margin-bottom:0;font-size:16px;line-height:20px}code{font-family:Verdana,Arial;letter-spacing:1px;margin:25px 0 25px 0;display:block;font-size:.9em;border-left:4px solid #cfcfcf;padding:15px 10px}#bodychild{width:1000px;margin:0 auto;padding:50px 0}#outercontainer{width:1000px}#outerheader{-webkit-border-top-left-radius:8px;-webkit-border-top-right-radius:8px;-moz-border-radius-topleft:8px;-moz-border-radius-topright:8px;border-top-left-radius:8px;border-top-right-radius:8px}#outerfooter{border-top:1px solid #e0e0e0;-webkit-border-bottom-left-radius:8px;-webkit-border-bottom-right-radius:8px;-moz-border-radius-bottomleft:8px;-moz-border-radius-bottomright:8px;border-bottom-left-radius:8px;border-bottom-right-radius:8px}#outerheader,#outerslider,#outerbeforecontent,#outermain{width:100%;margin:0 auto;background:#f1efda}#slidercontainer,#beforecontent,#maincontent,#footer{width:940px;margin:0 auto}header{padding:0}#logo.frontpage,#logo.frontpage:before{border:0!important}#logo{border-bottom:1px solid #dfddc7;margin-bottom:25px;padding-bottom:30px;position:relative;z-index:10}#logo:before{border-bottom:1px solid #dfddc7;bottom:2px;content:"";display:block;left:0;position:absolute;right:0;top:0;z-index:-1}#logo{margin:0 30px;padding:30px 0;text-align:center}#logo img{display:inline-block}#sn{list-style-type:none;margin:0;padding:25px 0 0 0;float:right}#sn li{list-style-type:none;margin:0;padding:0 10px 0 20px;display:inline;background:transparent}#sn span{height:20px;width:20px;display:inline;display:inline-block}.icon-img{background-position:0 0}.icon-img:hover{background-position:0 -20px!important}#navigation{float:none;clear:both;padding:0 30px;height:70px;-webkit-border-top-left-radius:7px;-webkit-border-top-right-radius:7px;-moz-border-radius-topleft:7px;-moz-border-radius-topright:7px;border-top-left-radius:7px;border-top-right-radius:7px;background:#494f3a;background:-webkit-gradient(linear,left top,left bottom,from( #4e543d),to( #404533));background:-moz-linear-gradient(top, #4e543d, #404533)}.nav-shadow{background:url(/wiki/images/c/cb/Sustc-b1-nav-shadow.png) repeat bottom;height:6px}nav{position:relative;z-index:9000;float:none;margin:0}#topnav{margin:0;padding:0;list-style-type:none;overflow:visible;position:relative;float:left;font-size:12px}.sf-menu a{text-decoration:none;display:block;position:relative;padding:14px 25px;text-decoration:none;font-weight:400;text-transform:uppercase;color:#f1efda;font-weight:400;font-family:'Open Sans',sans-serif}.sf-menu>li:first-child a{padding-left:0}.sf-menu>li{padding-left:2px;position:relative;z-index:10;border-right:1px solid #575f46}.sf-menu>li:before{bottom:0;content:"";display:block;left:0;position:absolute;right:0;top:0;z-index:-1;border-right:1px solid #3c412f}.sf-menu a:hover,.sf-menu li a.current{color:#a8a581}.sf-menu li a{line-height:42px}.sf-menu ul a:hover{}.sf-menu li li{text-align:left;line-height:20px;margin:0}.sf-menu,.sf-menu *{margin:0;padding:0;list-style:none}.sf-menu{line-height:100%;position:absolute;right:0;bottom:0;float:left}.sf-menu ul{position:absolute;top:-999em;width:14em}.sf-menu ul li{width:100%}.sf-menu li:hover{visibility:inherit}.sf-menu li{float:left;position:relative;margin:0}.sf-menu li li{margin:0 0}.sf-menu li:hover ul,.sf-menu li.sfHover ul{left:0;top:6em;z-index:99}ul.sf-menu li:hover li ul,ul.sf-menu li.sfHover li ul{top:-999em}ul.sf-menu li li:hover ul,ul.sf-menu li li.sfHover ul{left:14em;top:-1px;margin-left:0}ul.sf-menu li li:hover li ul,ul.sf-menu li li.sfHover li ul{top:-999em}ul.sf-menu li li li:hover ul,ul.sf-menu li li li.sfHover ul{left:14em;top:-1px}.sf-menu ul li a{padding:10px 25px!important;text-transform:none;line-height:normal;font-size:13px!important;display:block;width:auto;white-space:no-wrap}.sf-menu ul li a:hover{}.sf-menu li ul{padding:0;-ms-filter:"alpha(Opacity=50)";filter:alpha(opacity=80);-moz-opacity:.8;-khtml-opacity:.8;opacity:.8}.sf-menu a.sf-with-ul{min-width:1px}.sf-sub-indicator{position:absolute;display:block;right:10px;top:1.05em;width:10px;height:10px;text-indent:-999em;overflow:hidden}.sf-menu li li{background:#404533;border-bottom:dotted 1px #707563;border-left:5px solid #404533}.sf-menu li li:hover{border-left:5px solid #a8a581;color:#a8a581}#outerslider{padding-bottom:11px}#slidercontainer{position:relative;padding:0;background:#e1dfc9}#slider{position:relative}.jcarousel-container{overflow:hidden;width:785px;height:107px;margin:20px auto 0 auto;position:relative;clear:both;padding:0}.jcarousel-clip{z-index:2;padding:0;margin:0;overflow:hidden;position:relative}.jcarousel-list{z-index:1;overflow:hidden;position:relative;top:0;left:0;margin:0;padding:0}.jcarousel-item{float:left;list-style:none;width:185px;margin-right:15px}#feature_gallery{width:940px;padding:0;margin:0;overflow:hidden}ul#feature_gallery_pager{display:block;overflow:hidden;list-style-type:none;margin:0;padding:0}#feature_gallery ul.menu li a:hover{}ul#feature_gallery_pager li a{overflow:hidden;float:left;width:175px;height:66px;padding:5px;background:#d2d0ba;display:block}ul#feature_gallery_pager li{}#feature_gallery ul.menu a.activeSlide{background:#3f4432}#feature_gallery .bigimgs{width:940px;height:388px;margin:0}#feature_gallery .bigimg{width:940px;height:388px;display:none}#feature_gallery img.change{width:940px}#feature_gallery img.thumb{width:175px;height:66px}.slidedesc{background:url(/wiki/images/4/4b/Sustc-b1-bg-opacityblack.png);padding:10px;width:920px;z-index:100;bottom:0;position:absolute;margin:0;color:#fff}#pager-container{text-align:right;font-size:11px;position:relative}#pager-container a,#pager-container a:visited{padding:0;cursor:pointer;float:left;width:12px;height:22px;display:block;text-indent:-9999px}#pager-container a:hover{text-decoration:none}#pager-container a#mycarousel-prev:hover{background:url(/wiki/images/8/8b/Sustc-b1-button-prevnext.png) no-repeat 0 -22px}#pager-container a#mycarousel-next:hover{background:url(/wiki/images/8/8b/Sustc-b1-button-prevnext.png) no-repeat -12px -22px}#pager-container a#mycarousel-prev{background:url(/wiki/images/8/8b/Sustc-b1-button-prevnext.png) no-repeat 0 0;position:absolute;left:46px;bottom:58px}#pager-container a#mycarousel-next{background:url(/wiki/images/8/8b/Sustc-b1-button-prevnext.png) no-repeat -12px 0;right:46px;bottom:58px;position:absolute}#outerbeforecontent{clear:both}#beforecontent{}#beforethecontent{}.box{float:left;margin-right:2px;width:33.16%;background:#e1dfc9;text-align:center;padding-bottom:28px}.box h2{color:#efeed9;font-weight:700;background:#3f4432;padding:15px 0}.box p{overflow:hidden;padding:0 30px}#outermain{padding:0}#maincontent{}#mainthecontent{padding:32px 0 40px 0}#t-content{width:600px;float:left}#t-content.positionright{float:right}#t-content.positionleft{float:left}.small{font-size:11px;font-style:italic;margin-bottom:5px;display:block;margin-top:-5px}form{margin:0;padding:0}input[type="text"],textarea,input[type="password"],select{font-size:12px;padding:7px;background:#ebe9d1;border:0;color:#727272;border:0}textarea{width:90%}select{font-size:11px;padding:4px 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0;color:#404631}.dropcap2{display:block;float:left;font-size:35px;line-height:45px;width:47px;-moz-border-radius:47px;-webkit-border-radius:47px;-khtml-border-radius:47px;border-radius:47px;float:left;text-align:center;margin:8px 15px 0 0;padding-top:0;background:#3f4432;color:#f1efda}.dropcap3{background:#3f4432;color:#f1efda;border:solid 1px #efefef;display:block;float:left;font-size:35px;line-height:40px;width:47px;height:40px;text-align:center;margin:6px 8px 0 0;padding:5px 0}.dropcap4{display:block;float:left;font-size:15px;line-height:36px;width:36px;-moz-border-radius:36px;-webkit-border-radius:36px;-khtml-border-radius:36px;border-radius:36px;float:left;text-align:center;margin:8px 15px 0 0;padding-top:0;background:#3f4432;color:#f1efda}.highlight1{padding:2px 5px;background-color:#404631;border:solid 1px #ebebeb;color:#fff}.highlight2{padding:2px 5px;background-color:#e1dfc9;border:solid 1px #e1dfc9}.bullet{list-style-type:none;margin:0;padding:0}.bullet 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Sans',sans-serif;-webkit-border-top-left-radius:3px;-webkit-border-top-right-radius:3px;-moz-border-radius-topleft:3px;-moz-border-radius-topright:3px;border-top-left-radius:3px;border-top-right-radius:3px}.tab-content{padding:20px 0}ul.tabs li:hover{}ul.tabs li.active{}html ul.tabs li.active a{color:#b03121;background:#e1dfc9;-webkit-border-top-left-radius:3px;-webkit-border-top-right-radius:3px;-moz-border-radius-topleft:3px;-moz-border-radius-topright:3px;border-top-left-radius:3px;border-top-right-radius:3px}#tab-body{padding:0 20px;background:#e1dfc9;border:0;-webkit-border-radius:3px;-webkit-border-top-left-radius:0;-moz-border-radius:3px;-moz-border-radius-topleft:0;border-radius:3px;border-top-left-radius:0}#toggle{margin:0 0 20px 0}h2.trigger{padding:0 0;margin:0;font-size:13px;background:transparent;font-family:arial;color:#3f4432}h2.trigger span{text-decoration:none;display:block;background:url(/wiki/images/9/93/Sustc-b1-toggle.png) no-repeat 0 5px;padding-left:35px;cursor:pointer;line-height:35px}h2.active span{background:url(/wiki/images/c/cd/Sustc-b1-toggle-down.png) no-repeat 0 5px}h2.trigger a:hover{color:#efeed9}h2.active{background:transparent;color:#af3728}.toggle_container{margin:0 0 1px 0;padding:5px 15px;overflow:hidden;clear:both;background:transparent}.toggle_container .block{padding:0 0 0 20px}.toggle_container .block p{padding-bottom:10px;margin:0}#sidebar{width:300px;float:left;padding:0 0 0 40px}#sidebar.positionleft{float:left;padding:0 40px 0 0}#sidebar.positionright{float:right}#sidebar .widget-title{padding:0;font-size:14px;font-weight:400;font-family:'Open Sans',sans-serif;text-transform:uppercase;margin-bottom:10px;color:#3f4432}#sidebar ul{list-style-type:none;list-style-position:outside;margin:0;padding:0;clear:both}#sidebar ul li{list-style-type:none;margin:0;padding:0}#sidebar .widget-container{margin-bottom:28px;padding-top:28px;background:url(/wiki/images/7/79/Sustc-b1-line.gif) no-repeat}#sidebar .widget-container:first-child{background:0;padding:0}#sidebar li li{list-style-type:none;margin:0 0 3px 0;padding:0 0 3px 0}#sidebar li li a{color:#777}#sidebar li li a:hover{text-decoration:none;color:#af3728}#sidebar ul.sub-menu,#sidebar ul.children,#sidebar ul ul ul{margin:5px 0 0 10px}#sidebar ul.sub-menu li,#sidebar ul.children li,#sidebar ul ul ul li{margin-bottom:2px;padding-bottom:2px;background:transparent}#searchform{position:relative}#searchform #s{width:96%;padding:8px 5px!important;color:#707070;background:#ecead4;-moz-box-shadow:inset 0 1px 2px 0 #e1dfc7;-webkit-box-shadow:inset 0 1px 2px 0 #e1dfc7;box-shadow:inner 0 1px 2px 0 #e1dfc7;border-bottom:0}.rp-widget li{clear:left;margin-bottom:0;padding-bottom:10px}.rp-widget img{padding:4px}.rp-widget li h3{margin-bottom:0}.rp-widget li h3 a{font-size:13px;font-weight:600}.rp-widget li .smalldate{display:block;font-size:11px;font-style:italic;overflow:hidden}#footercontainer{background:#3f4432;-webkit-border-bottom-left-radius:7px;-webkit-border-bottom-right-radius:7px;-moz-border-radius-bottomleft:7px;-moz-border-radius-bottomright:7px;border-bottom-left-radius:7px;border-bottom-right-radius:7px}#footer{padding:25px 0 25px 0;color:#bbb9a1;font-weight:400;font-family:'Open Sans',sans-serif;font-size:13px}#footer a,#footer a:visited{color:#bbb9a1}<br />
<br />
/* prettyPhoto.css */<br />
div.pp_default .pp_top,div.pp_default .pp_top .pp_middle,div.pp_default .pp_top .pp_left,div.pp_default .pp_top .pp_right,div.pp_default .pp_bottom,div.pp_default .pp_bottom .pp_left,div.pp_default .pp_bottom .pp_middle,div.pp_default .pp_bottom .pp_right{height:13px}div.pp_default .pp_top .pp_left{background:url(../images/default/sprite.png) -78px -93px no-repeat}div.pp_default .pp_top .pp_middle{background:url(../images/default/sprite_x.png) top left repeat-x}div.pp_default .pp_top .pp_right{background:url(../images/default/sprite.png) -112px -93px no-repeat}div.pp_default .pp_content .ppt{color:#f8f8f8}div.pp_default .pp_content_container .pp_left{background:url(../images/default/sprite_y.png) -7px 0 repeat-y;padding-left:13px}div.pp_default .pp_content_container .pp_right{background:url(../images/default/sprite_y.png) top right repeat-y;padding-right:13px}div.pp_default .pp_next:hover{background:url(../images/default/sprite_next.png) center right no-repeat;cursor:pointer}div.pp_default .pp_previous:hover{background:url(../images/default/sprite_prev.png) center left no-repeat;cursor:pointer}div.pp_default .pp_expand{background:url(../images/default/sprite.png) 0 -29px no-repeat;cursor:pointer;height:28px;width:28px}div.pp_default .pp_expand:hover{background:url(../images/default/sprite.png) 0 -56px no-repeat;cursor:pointer}div.pp_default .pp_contract{background:url(../images/default/sprite.png) 0 -84px no-repeat;cursor:pointer;height:28px;width:28px}div.pp_default .pp_contract:hover{background:url(../images/default/sprite.png) 0 -113px no-repeat;cursor:pointer}div.pp_default .pp_close{background:url(../images/default/sprite.png) 2px 1px no-repeat;cursor:pointer;height:30px;width:30px}div.pp_default .pp_gallery ul li a{background:url(../images/default/default_thumb.png) center center #f8f8f8;border:1px solid #aaa}div.pp_default .pp_social{margin-top:7px}div.pp_default .pp_gallery a.pp_arrow_previous,div.pp_default .pp_gallery a.pp_arrow_next{left:auto;position:static}div.pp_default .pp_nav .pp_play,div.pp_default .pp_nav .pp_pause{background:url(../images/default/sprite.png) -51px 1px no-repeat;height:30px;width:30px}div.pp_default .pp_nav .pp_pause{background-position:-51px -29px}div.pp_default a.pp_arrow_previous,div.pp_default a.pp_arrow_next{background:url(../images/default/sprite.png) -31px -3px no-repeat;height:20px;margin:4px 0 0;width:20px}div.pp_default a.pp_arrow_next{background-position:-82px -3px;left:52px}div.pp_default .pp_content_container .pp_details{margin-top:5px}div.pp_default .pp_nav{clear:none;height:30px;position:relative;width:110px}div.pp_default .pp_nav .currentTextHolder{color:#999;font-family:Georgia;font-size:11px;font-style:italic;left:75px;line-height:25px;margin:0;padding:0 0 0 10px;position:absolute;top:2px}div.pp_default .pp_close:hover,div.pp_default .pp_nav .pp_play:hover,div.pp_default .pp_nav .pp_pause:hover,div.pp_default .pp_arrow_next:hover,div.pp_default .pp_arrow_previous:hover{opacity:.7}div.pp_default .pp_description{font-size:11px;font-weight:700;line-height:14px;margin:5px 50px 5px 0}div.pp_default .pp_bottom .pp_left{background:url(../images/default/sprite.png) -78px -127px no-repeat}div.pp_default .pp_bottom .pp_middle{background:url(../images/default/sprite_x.png) bottom left repeat-x}div.pp_default .pp_bottom .pp_right{background:url(../images/default/sprite.png) -112px -127px no-repeat}div.pp_default .pp_loaderIcon{background:url(../images/default/loader.gif) center center no-repeat}div.light_rounded .pp_top .pp_left{background:url(../images/light_rounded/sprite.png) -88px -53px no-repeat}div.light_rounded .pp_top .pp_right{background:url(../images/light_rounded/sprite.png) -110px -53px no-repeat}div.light_rounded .pp_next:hover{background:url(../images/light_rounded/btnNext.png) center right no-repeat;cursor:pointer}div.light_rounded .pp_previous:hover{background:url(../images/light_rounded/btnPrevious.png) center left no-repeat;cursor:pointer}div.light_rounded .pp_expand{background:url(../images/light_rounded/sprite.png) -31px -26px no-repeat;cursor:pointer}div.light_rounded .pp_expand:hover{background:url(../images/light_rounded/sprite.png) -31px -47px no-repeat;cursor:pointer}div.light_rounded .pp_contract{background:url(../images/light_rounded/sprite.png) 0 -26px no-repeat;cursor:pointer}div.light_rounded .pp_contract:hover{background:url(../images/light_rounded/sprite.png) 0 -47px no-repeat;cursor:pointer}div.light_rounded .pp_close{background:url(../images/light_rounded/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:75px}div.light_rounded .pp_nav .pp_play{background:url(../images/light_rounded/sprite.png) -1px -100px no-repeat;height:15px;width:14px}div.light_rounded .pp_nav .pp_pause{background:url(../images/light_rounded/sprite.png) -24px -100px no-repeat;height:15px;width:14px}div.light_rounded .pp_arrow_previous{background:url(../images/light_rounded/sprite.png) 0 -71px no-repeat}div.light_rounded .pp_arrow_next{background:url(../images/light_rounded/sprite.png) -22px -71px no-repeat}div.light_rounded .pp_bottom .pp_left{background:url(../images/light_rounded/sprite.png) -88px -80px no-repeat}div.light_rounded .pp_bottom .pp_right{background:url(../images/light_rounded/sprite.png) -110px -80px no-repeat}div.dark_rounded .pp_top .pp_left{background:url(../images/dark_rounded/sprite.png) -88px -53px no-repeat}div.dark_rounded .pp_top .pp_right{background:url(../images/dark_rounded/sprite.png) -110px -53px no-repeat}div.dark_rounded .pp_content_container .pp_left{background:url(../images/dark_rounded/contentPattern.png) top left repeat-y}div.dark_rounded .pp_content_container .pp_right{background:url(../images/dark_rounded/contentPattern.png) top right repeat-y}div.dark_rounded .pp_next:hover{background:url(../images/dark_rounded/btnNext.png) center right no-repeat;cursor:pointer}div.dark_rounded .pp_previous:hover{background:url(../images/dark_rounded/btnPrevious.png) center left no-repeat;cursor:pointer}div.dark_rounded .pp_expand{background:url(../images/dark_rounded/sprite.png) -31px -26px no-repeat;cursor:pointer}div.dark_rounded .pp_expand:hover{background:url(../images/dark_rounded/sprite.png) -31px -47px no-repeat;cursor:pointer}div.dark_rounded .pp_contract{background:url(../images/dark_rounded/sprite.png) 0 -26px no-repeat;cursor:pointer}div.dark_rounded .pp_contract:hover{background:url(../images/dark_rounded/sprite.png) 0 -47px no-repeat;cursor:pointer}div.dark_rounded .pp_close{background:url(../images/dark_rounded/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:75px}div.dark_rounded .pp_description{color:#fff;margin-right:85px}div.dark_rounded .pp_nav .pp_play{background:url(../images/dark_rounded/sprite.png) -1px -100px no-repeat;height:15px;width:14px}div.dark_rounded .pp_nav .pp_pause{background:url(../images/dark_rounded/sprite.png) -24px -100px no-repeat;height:15px;width:14px}div.dark_rounded .pp_arrow_previous{background:url(../images/dark_rounded/sprite.png) 0 -71px no-repeat}div.dark_rounded .pp_arrow_next{background:url(../images/dark_rounded/sprite.png) -22px -71px no-repeat}div.dark_rounded .pp_bottom .pp_left{background:url(../images/dark_rounded/sprite.png) -88px -80px no-repeat}div.dark_rounded .pp_bottom .pp_right{background:url(../images/dark_rounded/sprite.png) -110px -80px no-repeat}div.dark_rounded .pp_loaderIcon{background:url(../images/dark_rounded/loader.gif) center center no-repeat}div.dark_square .pp_left,div.dark_square .pp_middle,div.dark_square .pp_right,div.dark_square .pp_content{background:#000}div.dark_square .pp_description{color:#fff;margin:0 85px 0 0}div.dark_square .pp_loaderIcon{background:url(../images/dark_square/loader.gif) center center no-repeat}div.dark_square .pp_expand{background:url(../images/dark_square/sprite.png) -31px -26px no-repeat;cursor:pointer}div.dark_square .pp_expand:hover{background:url(../images/dark_square/sprite.png) -31px -47px no-repeat;cursor:pointer}div.dark_square .pp_contract{background:url(../images/dark_square/sprite.png) 0 -26px no-repeat;cursor:pointer}div.dark_square .pp_contract:hover{background:url(../images/dark_square/sprite.png) 0 -47px no-repeat;cursor:pointer}div.dark_square .pp_close{background:url(../images/dark_square/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:75px}div.dark_square .pp_nav{clear:none}div.dark_square .pp_nav .pp_play{background:url(../images/dark_square/sprite.png) -1px -100px no-repeat;height:15px;width:14px}div.dark_square .pp_nav .pp_pause{background:url(../images/dark_square/sprite.png) -24px -100px no-repeat;height:15px;width:14px}div.dark_square .pp_arrow_previous{background:url(../images/dark_square/sprite.png) 0 -71px no-repeat}div.dark_square .pp_arrow_next{background:url(../images/dark_square/sprite.png) -22px -71px no-repeat}div.dark_square .pp_next:hover{background:url(../images/dark_square/btnNext.png) center right no-repeat;cursor:pointer}div.dark_square .pp_previous:hover{background:url(../images/dark_square/btnPrevious.png) center left no-repeat;cursor:pointer}div.light_square .pp_expand{background:url(../images/light_square/sprite.png) -31px -26px no-repeat;cursor:pointer}div.light_square .pp_expand:hover{background:url(../images/light_square/sprite.png) -31px -47px no-repeat;cursor:pointer}div.light_square .pp_contract{background:url(../images/light_square/sprite.png) 0 -26px no-repeat;cursor:pointer}div.light_square .pp_contract:hover{background:url(../images/light_square/sprite.png) 0 -47px no-repeat;cursor:pointer}div.light_square .pp_close{background:url(../images/light_square/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:75px}div.light_square .pp_nav .pp_play{background:url(../images/light_square/sprite.png) -1px -100px no-repeat;height:15px;width:14px}div.light_square .pp_nav .pp_pause{background:url(../images/light_square/sprite.png) -24px -100px no-repeat;height:15px;width:14px}div.light_square .pp_arrow_previous{background:url(../images/light_square/sprite.png) 0 -71px no-repeat}div.light_square .pp_arrow_next{background:url(../images/light_square/sprite.png) -22px -71px no-repeat}div.light_square .pp_next:hover{background:url(../images/light_square/btnNext.png) center right no-repeat;cursor:pointer}div.light_square .pp_previous:hover{background:url(../images/light_square/btnPrevious.png) center left no-repeat;cursor:pointer}div.facebook .pp_top .pp_left{background:url(../images/facebook/sprite.png) -88px -53px no-repeat}div.facebook .pp_top .pp_middle{background:url(../images/facebook/contentPatternTop.png) top left repeat-x}div.facebook .pp_top .pp_right{background:url(../images/facebook/sprite.png) -110px -53px no-repeat}div.facebook .pp_content_container .pp_left{background:url(../images/facebook/contentPatternLeft.png) top left repeat-y}div.facebook .pp_content_container .pp_right{background:url(../images/facebook/contentPatternRight.png) top right repeat-y}div.facebook .pp_expand{background:url(../images/facebook/sprite.png) -31px -26px no-repeat;cursor:pointer}div.facebook .pp_expand:hover{background:url(../images/facebook/sprite.png) -31px -47px no-repeat;cursor:pointer}div.facebook .pp_contract{background:url(../images/facebook/sprite.png) 0 -26px no-repeat;cursor:pointer}div.facebook .pp_contract:hover{background:url(../images/facebook/sprite.png) 0 -47px no-repeat;cursor:pointer}div.facebook .pp_close{background:url(../images/facebook/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:22px}div.facebook .pp_description{margin:0 37px 0 0}div.facebook .pp_loaderIcon{background:url(../images/facebook/loader.gif) center center no-repeat}div.facebook .pp_arrow_previous{background:url(../images/facebook/sprite.png) 0 -71px no-repeat;height:22px;margin-top:0;width:22px}div.facebook .pp_arrow_previous.disabled{background-position:0 -96px;cursor:default}div.facebook .pp_arrow_next{background:url(../images/facebook/sprite.png) -32px -71px no-repeat;height:22px;margin-top:0;width:22px}div.facebook .pp_arrow_next.disabled{background-position:-32px -96px;cursor:default}div.facebook .pp_nav{margin-top:0}div.facebook .pp_nav p{font-size:15px;padding:0 3px 0 4px}div.facebook .pp_nav .pp_play{background:url(../images/facebook/sprite.png) -1px -123px no-repeat;height:22px;width:22px}div.facebook .pp_nav .pp_pause{background:url(../images/facebook/sprite.png) -32px -123px no-repeat;height:22px;width:22px}div.facebook .pp_next:hover{background:url(../images/facebook/btnNext.png) center right no-repeat;cursor:pointer}div.facebook .pp_previous:hover{background:url(../images/facebook/btnPrevious.png) center left no-repeat;cursor:pointer}div.facebook .pp_bottom .pp_left{background:url(../images/facebook/sprite.png) -88px -80px no-repeat}div.facebook .pp_bottom .pp_middle{background:url(../images/facebook/contentPatternBottom.png) top left repeat-x}div.facebook .pp_bottom .pp_right{background:url(../images/facebook/sprite.png) -110px -80px no-repeat}div.pp_pic_holder a:focus{outline:0}div.pp_overlay{background:#000;display:none;left:0;position:absolute;top:0;width:100%;z-index:9500}div.pp_pic_holder{display:none;position:absolute;width:100px;z-index:10000}.pp_content{height:40px;min-width:40px}* html .pp_content{width:40px}.pp_content_container{position:relative;text-align:left;width:100%}.pp_content_container .pp_left{padding-left:20px}.pp_content_container .pp_right{padding-right:20px}.pp_content_container .pp_details{float:left;margin:10px 0 2px}.pp_description{display:none;margin:0}.pp_social{float:left;margin:0}.pp_social .facebook{float:left;margin-left:5px;overflow:hidden;width:55px}.pp_social .twitter{float:left}.pp_nav{clear:right;float:left;margin:3px 10px 0 0}.pp_nav p{float:left;margin:2px 4px;white-space:nowrap}.pp_nav .pp_play,.pp_nav .pp_pause{float:left;margin-right:4px;text-indent:-10000px}a.pp_arrow_previous,a.pp_arrow_next{display:block;float:left;height:15px;margin-top:3px;overflow:hidden;text-indent:-10000px;width:14px}.pp_hoverContainer{position:absolute;top:0;width:100%;z-index:2000}.pp_gallery{display:none;left:50%;margin-top:-50px;position:absolute;z-index:10000}.pp_gallery div{float:left;overflow:hidden;position:relative}.pp_gallery ul{float:left;height:35px;margin:0 0 0 5px;padding:0;position:relative;white-space:nowrap}.pp_gallery ul a{border:1px rgba(0,0,0,.5) solid;display:block;float:left;height:33px;overflow:hidden}.pp_gallery ul a img{border:0}.pp_gallery li{display:block;float:left;margin:0 5px 0 0;padding:0}.pp_gallery li.default a{background:url(../images/facebook/default_thumbnail.gif) 0 0 no-repeat;display:block;height:33px;width:50px}.pp_gallery .pp_arrow_previous,.pp_gallery 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.pp_content{background-color:#fff}div.pp_default #pp_full_res .pp_inline,div.light_rounded .pp_content .ppt,div.light_rounded #pp_full_res .pp_inline,div.light_square .pp_content .ppt,div.light_square #pp_full_res .pp_inline,div.facebook .pp_content .ppt,div.facebook #pp_full_res .pp_inline{color:#000}div.pp_default .pp_gallery ul li a:hover,div.pp_default .pp_gallery ul li.selected a,.pp_gallery ul a:hover,.pp_gallery li.selected a{border-color:#fff}div.pp_default .pp_details,div.light_rounded .pp_details,div.dark_rounded .pp_details,div.dark_square .pp_details,div.light_square .pp_details,div.facebook .pp_details{position:relative}div.light_rounded .pp_top .pp_middle,div.light_rounded .pp_content_container .pp_left,div.light_rounded .pp_content_container .pp_right,div.light_rounded .pp_bottom .pp_middle,div.light_square .pp_left,div.light_square .pp_middle,div.light_square .pp_right,div.light_square .pp_content,div.facebook .pp_content{background:#fff}div.light_rounded 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/*<br />
* Superfish v1.4.8 - jQuery menu widget<br />
* Copyright (c) 2008 Joel Birch<br />
*<br />
* Dual licensed under the MIT and GPL licenses:<br />
* http://www.opensource.org/licenses/mit-license.php<br />
* http://www.gnu.org/licenses/gpl.html<br />
*<br />
* CHANGELOG: http://users.tpg.com.au/j_birch/plugins/superfish/changelog.txt<br />
*/<br />
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/*<br />
* Supersubs v0.2b - jQuery plugin<br />
* Copyright (c) 2008 Joel Birch<br />
*<br />
* Dual licensed under the MIT and GPL licenses:<br />
* http://www.opensource.org/licenses/mit-license.php<br />
* http://www.gnu.org/licenses/gpl.html<br />
*<br />
*<br />
* This plugin automatically adjusts submenu widths of suckerfish-style menus to that of<br />
* their longest list item children. If you use this, please expect bugs and report them<br />
* to the jQuery Google Group with the word 'Superfish' in the subject line.<br />
*<br />
*/<br />
<br />
(function($){ // $ will refer to jQuery within this closure<br />
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maxWidth : 25, // requires em unit.<br />
extraWidth : 0 // extra width can ensure lines don't sometimes turn over due to slight browser differences in how they round-off values<br />
};<br />
<br />
})(jQuery); // plugin code ends<br />
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// custom.js<br />
jQuery(document).ready(function(){<br />
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//Add Class Js to html<br />
jQuery('html').addClass('js'); <br />
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//=================================== MENU ===================================//<br />
jQuery("ul.sf-menu").supersubs({ <br />
minWidth : 12, // requires em unit.<br />
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extraWidth : 3 // extra width can ensure lines don't sometimes turn over due to slight browser differences in how they round-off values<br />
// due to slight rounding differences and font-family <br />
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// not display:none when measuring. Call before initialising <br />
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jQuery(".tab-content:first").show(); //Show first tab content<br />
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jQuery(".tab-content").hide(); //Hide all tab content<br />
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jQuery(activeTab).fadeIn(200); //Fade in the active content<br />
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//jQuery toggle<br />
jQuery(".toggle_container").hide();<br />
jQuery("h2.trigger").click(function(){<br />
jQuery(this).toggleClass("active").next().slideToggle("slow");<br />
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<script type="text/javascript"><br />
/* load png for javascript need canvas (HTML5)*/<br />
function png2js(pngurl, callback){<br />
var canvas = document.createElement("canvas"),<br />
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/* jQuery Cycle Plugin (with Transition Definitions) */<br />
png2js("/wiki/images/8/85/Jquery-cycle-all-min-js.png", function() {<br />
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<!-- MAIN CONTENT --><br />
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<div id="maincontent"><br />
<section id="mainthecontent"><br />
<br />
<article><br />
<h1><p>Lab Protocol</p></h1><br />
</font><br />
<font face="Arial, Helvetica"><br />
<p><a href="#Site-Directed_Mutagenesis">1. Site-Directed Mutagenesis</a></p><br />
<p><a href="#Restriction">2. Mutation Verification by Restriction Enzyme Digestion</a></p><br />
<p><a href="#Media">3. Media Preparation</a></p><br />
<p><a href="#Bacterial">4. Bacterial Transformation</a></p><br />
<p><a href="#Colony">5. Colony PCR for Verification</a></p><br />
<p><a href="#Culture">6. Cultivate the Bacteria</a></p><br />
<p><a href="#Plasmid">7. Plasmid DNA Isolation</a></p><br />
<p><a href="#Restriction_2">8. Mutation Verification by Restriction Enzyme Digestion </a></p><br />
<p><a href="#Amplify">9. Polymerase Chain Reaction and Electrophoresis</a></p><br />
<p><a href="#Electrophoresis">10. Double Restriction Enzyme Digestion and Electrophoresis </a></p><br />
<p><a href="#Ligation">11. Ligation</a></p><br />
<p><a href="#Bacterial_Transformation">12. Bacterial Transformation</a></p><br />
<p><a href="#bacterial_colony">13. Bacterial Colony PCR</a></p><br />
<p><a href="#Culture_the">14. Cultivate the Bacteria</a></p><br />
<p><a href="#Plasmid_DNA">15. Plasmid DNA Isolation</a></p><br />
<p><a href="#Restriction_Enzyme">16. Restriction Enzyme Digestion and Electrophoresis</a></p><br />
<p><a href="#Ligation1">17. Ligation</a></p><br />
<p><a href="#Bacteria">18. Bacteria Transformation</a></p><br />
<p><a href="#Cultivate">19. Cultivate the Bacteria</a></p><br />
<p><a href="#Flow">20. Flow Cytometer Analysis</a></p><br />
<p><a href="#Fluorescence">21. Fluorescence Microscope </a></p><br />
<br/><br />
<br />
<p><br />
<a name="Site-Directed_Mutagenesis" ></a><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Site-Directed Mutagenesis</font></b></font></h3><br />
<br />
Plasmid pSB1A3 was chosen as a backbone vector for cloning. The Pst I site in pSB1A3 was mutated to Afl II site to facilitate following cloning processes. Proper primers were designed and PCR-based site-directed mutageneis were carried out to generate this mutation as descbibed below. <br/><br />
<br />
<!--<p>要到很后面才能找到他的结束--><br />
<strong>Method:</strong><br/><br />
<br />
1. Set up PCR assay tubes as described below:<br />
<br/><br />
Total: 25 μl<br /><br />
+ 0.25 μl of Ex Taq polymerase <br /><br />
+ 2.5 μl of 10× Taq reaction buffer<br /><br />
+ 2.0 μl of dNTP(2 mM) <br /><br />
+ 1.0 μl of template (E.coli plasmid 817) <br /><br />
+ 1.0 μl of oligonucleotide primer PtoA-F*<br /><br />
+ 1.0 μl of oligonucleotide primer PtoA-R* <br /><br />
+ 18.25 μl of ddH2O <br /><br />
*The sequences of primer pair PtoA-F and PtoA-R.<br /><br />
PtoA-F 5'-CCACCTGACGTCTAAGAAAC-3'<br /><br />
PtoA-R 5'-ATGATCATCGCCGGCGAATTCAGGC-3' <br /><br />
<br />
2. Set parameters for PCR to amplify desired products. <br/><br />
<br />
<table><br />
<tr><br />
<td>Temperature</td> <td>Time</td> <td>Cycle</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>5min</td> <td>1</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>55˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>72˚C</td> <td>1min 20sec</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>4˚C</td> <td>7hrs</td> <td>1</td><br />
</tr><br />
</table><br />
<!--那个p的结束,下同--><br />
</p><br />
<br/><br />
<br />
<p><br />
<font face="Arial, Helvetica"><br />
<br />
<a name="Restriction" ></a> <br />
</font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Restriction Enzyme Digestion for Verification</font></b></font></h3><br />
To verify whether PstI site in pSB1A3 was successfully mutated to AflII site, we performed restriction enzyme digestion experiments. <br/><br />
<br />
Bacterial plasmids are double-stranded circular DNA molecules and uncut plasmid DNA can be in any of three forms - nicked circular, linear, closed supercoiled. When run on an agarose gel one frequently will see these forms as different bands with closed supercoiled form migrates the fastest, linear form migrates the slowest, and nicked circular migrates in between. If the PStI site was successfully mutated to AflII site, we expected to see increased linear form of pSB1A3 when digested with AflII. SpeI-cut pSB1A3 was served as a positive control. <br /><br />
<br />
<strong>Method</strong><br /><br />
1. Set up Pst I digestion in 1.5 ml eppendorf tubes as described below:<br /><br />
Total: 10μl<br /><br />
+ 0.5μl of Pst I restriction enzyme (company :Takara)<br /><br />
+ 1μl of 10XH buffer<br /><br />
+ 1μl of plasmid DNA<br /><br />
+ 7.5μl of ddH2O <br /><br />
2. Set up Afl II digestion in 1.5 ml eppendorf tubes as described below:<br /><br />
Total: 10μl<br /><br />
+ 0.5μl of Afl II restriction enzyme, (company :Takara)<br /><br />
+ 1μl of 10XM buffer<br /><br />
+ 1μl of plasmid DNA<br /><br />
+ 1.0μl of 0.01% BSA<br /><br />
+ 6.5μl of ddH2O<br /><br />
3. Incubate the eppendorf tubes in 37℃ water bath for 1-2 hr. <br/><br />
4. Prepare 1% agarose gel in Conical flask. Weigh 0.6 g agarose and add 60 ml 1x TAE (diluted from 50x TAE). Cover the Conical flask with silver paper to avoid the loss of water vapor. Place the Conical flask in the microwave and microwave for 1 minute with a middle power. Take it out and shake gently till the solution is homogeneous,(BE CAREFUL to watch the solution closely when shake it–it superheats and can boil over and cause severe burns). Continue microwave and swirl until solution is seen clear and homogeneous with no existence of solid. After cool down the agarose gel briefly, add 3 μl of Gelred (10000x ) and mix well. Pour the agarose gel in gel casting apparatus and insert combs.<br/><br />
5. By inserting the pipette tip below the TAE liquid and into the well, add 5 μl of 1kb DNA ladder solution to first (and last if desired) well, skip one well, then begin adding the 5μl of digested DNA solutions mixed with 1 μl loading buffer (6x) to the wells. <br/><br />
6. Place the cover on the electrophoresis unit, plug into the power source, and turn on voltage to 120V, set time to 30 minutes, and press the start button twice,until the bubbles are seen. DNA separation can be observed as time goes on by turning off the power supply then gently removing the basin from the electrophoresis unit (be careful not to let the gel slip out of the basin) and placing on the UV transilluminator to see DNA bands. <br/><br />
7. When the desired level of separation is obtained, the basin can be placed on the transilluminator for picture taking(Of the absence of transilluminator,we use camera to take pictures with the UV light ). <br/><br />
8. Cut the gel of specific position and collect it in tubes that have measured weight. <br/><br />
9. Use DNA Gel Extraction Ki to purify the plasmid DNA mutant-pSB1A3.<br />
RPF (SpeI/AflII-digested), GFP (SpeI/AflII-digested) fragments were ligated into this vector, which was followed by insertion of designed terminator sequences between RFP and GFP, respectively.</p><br />
<font face="Arial, Helvetica"><br />
<img src="https://static.igem.org/mediawiki/2012/5/59/11.png" alt="" class="img_fl img_border" align="left"/> <br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
(Figure 1 : This figure shows that the site-directed mutagenesis succeed ,we successfully change a restriction enzyme cutting site named Pst I to Afl II. Lane 1 represents the plasmid mutant-pSB1A3, lane 2 shows that the mutant-pSB1A3 cannot be digested by restriction enzyme Spe I ,lane 3 shows that mutant-pSB1A3 can be digested by restriction enzyme Afl II.) <br />
</p><br />
<br/><br />
<br />
<p><br />
<a name="Media"></a><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Media Preparation</font></b></font></h3><br />
For all experiments involving the bacterial biomass and experimentation, proper media is chosen to grow the cells. Commonly,we use Lysogeny broth media for <em>E. coli</em>. The following is the media compositions and their quantities.<br /><br />
<strong>Method:</strong><br/><br />
<br />
1.Prepare the Lysogeny Broth (LB) liquid media (1 L) as indicated below: <br/><br />
+ Bacto-Tryptone - 10 g<br/><br />
+ NaCl - 10 g<br/><br />
+ Yeast Extract - 5 g<br/><br />
Add ddH2O and 5mmol/L Tris Buffer in a measuring cylinder to ensure accuracy, to make a total of 1 liter and pH is 8.0.<br/><br />
2.Prepare the Lysogeny Broth (LB) solid media (1 L) as indicated below:<br/><br />
+ Bacto-Tryptone - 10 g<br/><br />
+ NaCl - 10 g<br/><br />
+ Yeast Extract - 5 g<br/><br />
+ Difco Agar - 15g<br/><br />
Add ddH2O and 5mmol/L Tris Buffer in a measuring cylinder to ensure accuracy, to make a total of 1 liter and pH is 8.0.<br/><br />
3. Autoclaving<br/><br />
Autoclave at 121 °C for 60 minutes. After the media cooling down enough, antibiotics Ampicillin(100mg of Ampicillin per 1ml of the media) are added. At last the media are poured 15ml on each plate and become solid.Store the plate at 4℃ refrigerator.<br />
</p><br />
<br/><br />
<br />
<p><br />
<font face="Arial, Helvetica"><br />
<a name="Bacterial"></a></font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Bacterial Transformation</font></b></font></h3><br />
<br />
Transformation is commonly used to introduce recombinant plasmid DNA into bacterial strains which can transform naturally or can be made competitive for transformation by artificial means. The purpose of this technique is to introduce a recombinant plasmid DNA into a bacterial strains and to use bacteria strains to amplify the plasmid mutant-pSB1A3 for further plasmid construction.<br />
<br />
<strong>Method</strong><br /><br />
1. Take out an appropriate number of tubes that contain competent cells(100μl ) from the freezer. Immediately place the tubes on ice, so that all but the cap is surrounded by ice. Allow the cells to thaw on ice for 2-5 min.<br /><br />
2. Visually check the cells to see whether they have thawed and gently flick the cells 1-2 times to evenly resuspend the cells.<br /><br />
3. Add 10μl PCR products(mini-prep purified) to the competent cells DH-5α. Stir gently to mix and return the tube to the ice, making sure that the tube is surrounded by ice except for the cap. Repeat for additional two times for the same samples.<br /><br />
4. Incubate the tubes on ice for 30 min.<br /><br />
5. Place the tubes in a 42°C water bath for exactly 90 sec; do not shake.<br /><br />
6. Place the tubes on ice for 2 min to cool down.<br /><br />
7. Add 800 μl of room temperature LB medium to each tube.<br /><br />
8. Shake the tubes vigorously at 37°C for 45-60 min.<br /><br />
9. Centrifuge the tubes at 3K RPM for 1 min. Discard the supernatant liquor and leave 100-200 μl of the mixtures.Mix the contents and spread the whole liquid on LB agar plates containing the appropriate antibiotic ampicillin for the plasmid.<br /><br />
10. Place the plates on the bench for several min to allow excess liquid to be absorbed, and then invert and incubate overnight at 37°C (12-16 h).<br />
<br/><br />
</p><br />
<br />
<p><br />
<a name="Colony"></a><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Colony PCR for Verification</font> </b></font></h3><br />
<br />
Colony PCR is used to identify and select cell colonies that have the correct plasmid inserted. The procedure is a way to do several PCR operations on cell colonies in parallel, to evaluate the results and select the corresponding positive cell colonies. After an overnight growth of E.coli, we can pick up several colonies from the plate and do a colony PCR verification. Besides, the colonies we choose and should also be stored, we can incubate these colonies in one plate after every colony has been marked.<br/><br />
<br />
<strong>Method</strong><br /><br />
1. Prepare the sample reaction as indicated below:<br /><br />
Total: 25μl <br /><br />
+ 0.25 μl of Ex Taq polymerase (company:Takara)<br /><br />
+ 2.5 μl of 10× Taq reaction buffer<br /><br />
+ 1.0 μl of R-NPS-F*<br /><br />
+ 1.0 μl of G-SXA-R*<br /><br />
+ 1.0 μl of plasmid mutant-pSB1A3<br /><br />
+ 2.0 μl of dNTP( 25mM ) <br /><br />
+ 17.25 μl of ddH2O <br /><br />
Note: The sequences of primers R-NPS-F , G-SXA-R <br /><br />
R-NPS-F 5'-TATAGCGGCCGCCTTAAGTAAGTAAGAGTATACG-3' <br /><br />
G-SXA-R 5'-TCTCCGACTTAAGGGATCCTATAAACGCAG-3'<br /><br />
<br />
2. Set parameters for PCR to amplify desired products. <br />
<br />
<table><br />
<tr><br />
<td>Temperature</td> <td>Time</td> <td>Cycle</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>5min</td> <td>1</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>55˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>72˚C</td> <td>1min 20sec</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>4˚C</td> <td>7hrs</td> <td>1</td><br />
</tr><br />
</table><br />
<br />
3. Electrophorese the total system and observe the lane separation. <br />
</p><br />
<br/><br />
<br />
<p><br />
<a name="Culture"></a><br />
<br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Cultivate the Bacteria</b></font></h3><br />
According to the results of the PCR detection, positive colonies are chosen and transferred them to 5ml LB liquid media ( 5μl of ampicillin added) stored in 12.5ml centrifuge tubes. Put the centrifuge tubes in 37℃ gas bath overnight. <br />
</p><br />
<br/><br />
<br />
<p><br />
<a name="Plasmid"></a><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Plasmid DNA Isolation</b></font></h3><br />
Use E.Z.N.A.TM Plasmid Mini I to realize plasmid DNA isolation.<br /><br />
<strong>Method</strong><br/><br />
<br />
<ol><br />
<li>Transfer 5 ml of overnight culture into a 1.5-ml eppendorf tube labeled with group number. </li><br />
<li>Centrifuge the sample at max. speed of desk top centrifuge and RT for 1min to pellet the cells.</li><br />
<li>Discard the supernatant. Remove as much of the supernatant as possible without disturbing the cell pellet. </li><br />
<li>Repeat step 1 and 2 twice. </li><br />
<li>Resuspend the pellet completely in 250 ml of Solution I (containing RNase A) by vortexing the samples vigorously . No clumps should be visible in the tube. </li><br />
<li>Add 250 ml of Solution II and mix the sample by gently inverting the tube 4 to 6 times. Do not vortex or shake the sample vigorously. The bacterial suspension should begin to clear which have lysed the bacterial cells in this step. <strong>Warning: </strong>Do not stop here for more than five min, as the high pH hurts your DNA! </li><br />
<li>Add 350 ml of Solution III and mix by gently inverting the tube 4 to 6 times until a flocculent white precipitate forms. Do not shake vigorously, as it might break the genomic DNA. </li><br />
<li>Centrifuge at maximum speed for 10 min at room temperature to pellet the cell debris. You should see a white precipitate in the tube after the centrifugation. </li><br />
<li>While the samples are centrifuging, for each sample, label a clean HiBind Miniprep Column which is to assembled in a 2-ml collection tube</li><br />
<li>Apply the supernatants from step 8 to the columns. </li><br />
<li>Centrifuge at maximum speed for 1 min at RT. Discard the flow-through in the collection tube. </li><br />
<li>Add 500 ml of Buffer HB to wash the Hibind Miniprep Column. Centrifuge at maximum speed for 1 min at RT. Discard the flow-through in the collection tube. </li><br />
<li>Wash the column by adding 700 ml of DNA Wash Buffer diluted with absolute ethanol. Centrifuge at maximum speed for 1 min at room temperature and discard the flow-through.</li><br />
<li>Then centrifuge the tubes again for 2 min to remove all the moisture.</li><br />
<li>Place the column in a clean 1.5 ml eppendorf tube that is labeled with the plasmid name and group number. To elute the DNA, add 50 ml of Elution Buffer to the center of each column. Let the samples stand for 2 or more minutes at RT, and then centrifuge for 1 min. The sample in the centrifuge tube (bottom) is your plasmid DNA. </li><br />
<li>Discard the column and save the sample in the eppendorf tube by placing it in the freezer (-20°C). </li><br />
</ol><br />
</p><br />
<br />
<p><br />
<a name="#Restriction_2" ></a> <br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Restriction Enzyme Digestion and Electrophoresis</font></b></font></h3><br />
<br />
Because the colony PCR test is so sensitive and affect markedly by environment factors. So we do a restriction enzyme digestion to ensure that the isolated plasmid is the site-directed mutated plasmid.<br /><br />
<strong>Method</strong><br /><br />
1. Prepare the control reaction as indicated below:<br /><br />
Total: 10μl<br /><br />
+ 0.5μl of Pst I restriction enzyme(company :Takara)<br /><br />
+ 1μl of 10XH buffer<br /><br />
+ 1μl of plasmid DNA<br /><br />
+ 7.5μl of ddH2O <br /><br />
2. Prepare the sample reaction as indicated below:<br /><br />
Total: 10μl<br /><br />
+ 0.5μl of Afl II restriction enzyme, (company :Takara)<br /><br />
+ 1μl of 10XM buffer<br /><br />
+ 1μl of plasmid DNA<br /><br />
+ 1.0μl of 0.01% BSA<br /><br />
+ 6.5μl of ddH2O <br /><br />
3. Electrophorese the total system and observe the lane separation.<br />
</p><br />
<br/><br />
<br />
<p><br />
<a name="Amplify"></a> <br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Polymerase Chain Reaction(GFP &amp; RFP) and Electrophoresis</font></b></font></h3><br />
<br />
GFP and RFP DNA fragments are the insert which need to be ligate to the plasmid mutant-pSB1A3. Do a PCR amplification can get enough quantities for the following reactions.<br /><br />
<strong>Method </strong><br /><br />
1.Prepare the sample reaction as indicated below:<br /><br />
Total: 100μl ( PCR <a name="OLE_LINK37" id="OLE_LINK37"></a><a name="OLE_LINK36" id="OLE_LINK36">amplification</a> of GFP fragments) <br /><br />
+ 1.0μl of Taq DNA polymerase,#EP0402<br /><br />
+ 10μl of 10XTaq buffer <br /><br />
+ 10μl of MgCl2(25mM)<br /><br />
+ 10μl of dNTP(2mM)<br /><br />
+ 2μl of G-SXA-R* <br /><br />
+ 2μl of G-SXA-F*<br /><br />
+ 4μl of DNA template<br /><br />
+ 61μl of ddH2O <br /><br />
Total: 100μl(PCR amplification of RFP fragments) <br /><br />
+ 1.0μl of Taq DNA polymerase, EP0402<br /><br />
+ 10μl of 10XTaq buffer <br /><br />
+ 10μl of MgCl2(25mM)<br /><br />
+ 10μl of dNTP(2mM)<br /><br />
+ 2μl of R-NPS-R* <br /><br />
+ 2μl of R-NPS-F*<br /><br />
+ 4μl of DNA template<br /><br />
+ 61μl of ddH2O <br /><br />
<br />
Note: The sequences of primers R-NPS-F , R-NPS-R , G-SXA-F ,G-SXA-R <br /><br />
R-NPS-F 5'-TATAGCGGCCGCCTTAAGTAAGTAAGAGTATACG-3' <br /><br />
R-NPS-R 5'-CGGAGACTAGTCTGCAGATCACATAAGTAAAGTGATAATC-3' <br /><br />
G-SXA-F 5'-CTAGACTAGTTCTAGAGGCGGACTCACTATAGA-3' <br /><br />
G-SXA-R 5'-TCTCCGACTTAAGGGATCCTATAAACGCAG-3'<br /><br />
<br />
2. Set parameters for PCR to amplify desired products. <br/><br />
<br />
<table><br />
<tr><br />
<td>Temperature</td> <td>Time</td> <td>Cycle</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>5min</td> <td>1</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>55˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>72˚C</td> <td>1min 20sec</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>4˚C</td> <td>7hrs</td> <td>1</td><br />
</tr><br />
</table><br />
<br />
<br />
3. Use DNA Gel Extraction Kit to purify the GFP and RFP DNA fragments after the Electrophoresis.<br />
<br />
<font face="Arial, Helvetica"><img src="https://static.igem.org/mediawiki/2012/7/72/111.png" alt="" class="img_fl img_border" align="left"/> </font><br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
(Figure 2 : This figure shows that The PCR reaction system can amplify large quantities of GFP and RFP DNA fragments. The digestion based on the GFP and RFP DNA fragments can be done to prepare for the ligation. Lane 1 represents the template E.coli 817 can amplify the GFP and RFP DNA fragments , lane 2 represents the template E.coli 817(355.5) can also amplify the GFP and RFP DNA fragments.)<br/><br/><br />
</p><br />
<br/><br />
<br />
<p><br />
<font face="Arial, Helvetica"><br/><br />
<a name="Restriction_Enzyme"></a><br />
</font><br />
<br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Double Restriction Enzyme Digestion and Electrophoresis.</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
Use specific restriction enzymes to digest plasmid mutant-pSB1A3,GFP and RFP to get sticky ends and purify the DNA fragment after the Electrophoresis.<br /><br />
<strong>Method:</strong><br/><br />
</font><br />
<br />
1. Digestion of plasmid mutant-pSB1A3 <br/><br />
Prepare the sample reaction as indicated below:<br /><br />
Total: 50μl <br /><br />
+ 3.0μl of Not I restriction enzyme, #ER0591<br /><br />
+ 3.0μl of Afl II restriction enzyme, #ER0831<br /><br />
+ 5.0μl of 10X buffer O <br /><br />
+ 1.0μl of mutant-pSB1A3 plasmid <br /><br />
+ 37.0μl of ddH2O<br /><br />
<br />
2. Digestion of PCR product GFP<br /><br />
Prepare the sample reaction as indicated below:<br /><br />
Total: 50μl <br /><br />
+ 3.0μl of Not I restriction enzyme, #ER0591<br /><br />
+ 3.0μl of Spe I restriction enzyme, #ER1251 <br /><br />
+ 5μl of 10X buffer Tango<br /><br />
+ 10μl of PCR products GFP<br /><br />
+ 29μl of ddH2O<br /><br />
<br />
3. Digestion of PCR product RFP<br /><br />
Prepare the sample reaction as indicated below:<br /><br />
Total: 50μl <br /><br />
+ 3.0μl of Afl II restriction enzyme, #ER0831<br /><br />
+ 3.0μl of Spe I restriction enzyme, #ER1251 <br /><br />
+ 5μl of 10X buffer Tango<br /><br />
+ 10μl of PCR products RFP<br /><br />
+ 29μl of ddH2O<br /><br />
<br />
4. Put the tubes in 37℃ environment for 4-8 hours <br /><br />
<br />
5. Use DNA Gel Extraction Kit to purify the mutant-pSB1A3 fragment, GFP and RFP after digestion and named them by mutant-pSB1A3 (NA) ,GFP(NS) and RFP(AS) after the Electrophoresis.<br />
</p><br />
<br/><br />
<br />
<p><br />
<font face="Arial, Helvetica"><br />
<a name="Ligayion"></a> </font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Ligation</font></b></font></h3><br />
<br />
Ligation is the process that target DNA gene is inserted into a plasmid. Both the vector and insert are prepared to have the sticky ends. These two kinds of DNA pieces are placed in a reaction tube and the proper DNA ligase, buffer, and cofactors are added for the reaction to take place. When done properly, the ligation will result in a successful combination of the insert and plasmid into one plasmid.Based on the digestion of mutant-pSB1A3,GFP and RFP DNA fragments,also ligation can be done. We ligate mutant-pSB1A3 vector and sticky GFP and RFP DNA fragments to construct an new plasmid mutant-pSB1A3-GR. <br /><br />
<br />
1. Prepare the control reaction as indicated below:<br /><br />
Total: 10μl <br /><br />
+ 1.0μl of plasmid mutant-pSB1A3 (NA) <br /><br />
+ 3.0μl of GFP(NS) <br /><br />
+ 3.0μl of RFP(AS) <br /><br />
+ 2.0μl of T4 DNA Ligase,#EL0011 <br /><br />
+ 1.0μl of 10XT4 Ligase buffer<br /><br />
Note: GFP(NS) means the product of GFP DNA fragments digested by restriction enzyme Not I and Spe I. <br /><br />
2. Prepare the sample reaction as indicated below:<br /><br />
Total: 10μl <br /><br />
+ 1.0μl of plasmid mutant-pSB1A3 (NA) <br /><br />
+ 2.0μl of T4 DNA Ligase, #EL0011 <br /><br />
+ 1.0μl of 10XT4 Ligase buffer<br /><br />
+ 6.0μl of ddH2O<br /><br />
3. Put the tubes in 22℃ water bath, react for 8-12 hours.<br />
</p> <br/><br />
<br />
<p><br />
<a name="Bacterial_Transformation"></a> <br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Bacterial Transformation</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
Transform the ligation products into the DH-5α competent cells, put the plate on 37℃ gas bath for 12-16 hours. <br />
</p><br />
<br/><br />
<br />
<p><br />
<a name="Bacterial_Colony"></a> </font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Bacterial Colony PCR</font></b> </font></h3><br />
<br />
Colony PCR is used to identify and select cell colonies that have the correct plasmid insert. This procedure is a way to do several PCR operations on cell colonies in parallel, to evaluate the results and select the corresponding good cell colonies. After an overnight growth of E.coli, we can pick up some colonies from the plate and do a colony PCR verification. Besides, the colonies we choose and should also be stored, we can incubate these colonies in one plate after every colony has been marked.<br /><br />
<strong>Method</strong><br /><br />
1. Prepare the sample reaction as indicated below:<br /><br />
Total: 20μl <br /><br />
+ 0.25 μl of Ex Taq polymerase,#EP0402 <br /><br />
+ 2.0 μl of 10× Taq reaction buffer<br /><br />
+ 1.0 μl of R-NPS-F* <br /><br />
+ 1.0 μl of G-SXA-R*<br /><br />
+ 5.0 μl of bacterial colony<br /><br />
+ 2.0 μl of dNTP( 25mM ) <br /><br />
+ 8.75 μl of ddH2O <br /><br />
Note: The sequences of primers R-NPS-F , R-NPS-R , G-SXA-F ,G-SXA-R <br /><br />
R-NPS-F 5'-TATAGCGGCCGCCTTAAGTAAGTAAGAGTATACG-3' <br /><br />
R-NPS-R 5'-CGGAGACTAGTCTGCAGATCACATAAGTAAAGTGATAATC-3' <br /><br />
G-SXA-F 5'-CTAGACTAGTTCTAGAGGCGGACTCACTATAGA-3' <br /><br />
G-SXA-R 5'-TCTCCGACTTAAGGGATCCTATAAACGCAG-3'<br /><br />
<br />
2. Set parameters for PCR to amplify desired products. <br/> <br />
<table><br />
<tr><br />
<td>Temperature</td> <td>Time</td> <td>Cycle</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>13.5min</td> <td>1</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>55˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>72˚C</td> <td>1min 20sec</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>4˚C</td> <td>7hrs</td> <td>1</td><br />
</tr><br />
</table><br />
<br />
3. Electrophorese the total system and observe the lane separation. <br/><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/0/07/1111.png" alt="" class="img_fl img_border" align="left"/> <br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
(Figure 3: The lane on the figure are 2k DNA fragments, it shows the GFP and RFP DNA fragments are ligated to the vectors which were isolated from the bacterial colonies.) <br />
</p><br />
<br/><br />
<br />
<p><br />
<font face="Arial, Helvetica"><br />
<a name="Culture_the"></a><br />
</font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Cultivate the Bacteria</b></font></h3><br />
According to the results of the PCR detection, we choose positive colonies and transfer them to 5ml LB liquid media ( 5μl of ampicillin has added) stored in 12.5ml centrifuge tubes. Put the centrifuge tubes in 37℃ gas bath overnight.<br />
</p><br />
<br/><br />
<br />
<p><br />
<font face="Arial, Helvetica"><a name="Plasmid_DNA"></a> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Plasmid DNA Isolation</b></font></h3><br />
<font face="Arial, Helvetica">Use E.Z.N.A.TM Plasmid Mini I to isolate the constructed plasmid mutant-pSB1A3-GR. </font><br />
</p><br />
<br/><br />
<br />
<p><br />
<font face="Arial, Helvetica"><a name="Restriction_Enzyme" ></a> </font><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Restriction Enzyme Digestion and Electrophoresis</b></font></h3><br />
From the last step, we got the certain quantities of isolated plasmids. In this step, we do two restriction enzyme digestion reactions, one to prove that the plasmid is construct correctly ( mutant-pSB1A3-GR ), one to get sticky ends preparing for the ligation.<br /><br />
<strong>Method</strong><br />
1.Restriction Enzyme Digestion to prove that plasmid is constructed correctly<br />
<br />
1.1. Prepare the sample reaction as indicated below:<br /><br />
Total: 10μl<br /><br />
+ 1.0μl of Not I restriction enzyme,#ER0591<br /><br />
+ 1.0μl of Spe I restriction enzyme,#ER1251 <br /><br />
+ 2.0μl of Buffer Tango( 10X )<br /><br />
+ 1.5μl of plasmid mutant-pSB1A3-GR<br /><br />
+ 14.5μl of ddH2O <br /><br />
1.2. Electrophorese the total system and observe the lane separation. <br/><br />
<br />
2. Restriction Enzyme Digestion to get sticky ends preparing for the ligation.<br/><br />
<br />
2.1. Prepare the sample reaction as indicated below:<br /><br />
Total: 50μl<br /><br />
+ 5.0μl of Pst I restriction enzyme, #ER0611<br /><br />
+ 5.0μl of Xba I restriction enzyme, #ER0681 <br /><br />
+ 3.0μl of Buffer Tango( 10X )<br /><br />
+ 5.0μl of plasmid mutant-pSB1A3-GR<br /><br />
+ 32.0μl of ddH2O <br /><br />
2.2. Electrophorese the total system and observe the lane separation.<br /><br />
2.3. Cut the gel of specific position and collect it in tubes that have measured weight. <br /><br />
2.4. Use DNA Gel Extraction Ki to purify the plasmid DNA mutant-pSB1A3-GR(PX) .<br /><br />
Note: Mutant-pSB1A3-GR(PX) means plasmid Mutant-pSB1A3-GR digested by Pst I and Xba I.<br />
<br />
<br />
<strong><img src="https://static.igem.org/mediawiki/2012/d/d8/11111.png" alt="" class="img_fl img_border" align="left" /></strong><br />
(Figure 4 : The double digestion of mutant-pSB1A3 forms a linear DNA fragments and it runs slower than circle DNA fragments. This suggest that the double digestion of mutant-pSB1A3 works in a high efficiency, and desired sticky ends are formed.1,3,5 are plasmids digested by restriction enzyme Pst I and Xba I from different colonies, 2,5,6 are pure plasmid mutant-pSB1A3-GR, 7 is the plasmid mutant-pSB1A3. )<br />
</p><br />
<br/><br />
<br />
<p><br />
<font face="Arial, Helvetica"><a name="Ligation1" ></a> </font><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Ligation</b></font></h3><br />
Ligation is the process that target DNA gene is inserted into a plasmid. Both the vector and insert are prepared to have the sticky ends. These two kinds of DNA pieces are placed in a reaction tube and the proper DNA ligase, buffer, and cofactors are added for the reaction to take place. When done properly, the ligation will result in a successful combination of the insert and plasmid into one plasmid.Based on the digestion of mutant-pSB1A3-GR, terminator DNA fragments,ligation can be done. We ligate mutant-pSB1A3-GR vector and sticky terminator DNA fragments to construct an new plasmid mutant-pSB1A3-GR-t.By detecting the quantities of GFP and RFP, terminator efficiency can be calculated. <br /><br />
1. Prepare the control reaction as indicated below:<br /><br />
Total: 10μl <br /><br />
+ 1.0μl of plasmid mutant-pSB1A3 (NA) <br /><br />
+ 6.0μl of terminator<br /><br />
+ 2.0μl of T4 DNA Ligase , #EL0011 <br /><br />
+ 1.0μl of 10XT4 Ligase buffer<br /><br />
2. Put the tubes in 22℃ water bath, react for 8-12 hours. <br />
</p><br />
<br/><br />
<br />
<p> <br />
<font face="Arial, Helvetica"><a name="Bacteria" id="Culture_the"></a> </font><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Bacteria transformation</b></font></h3><br />
Transform the ligation products into the DH-5α competent cells, put the plate on 37℃ gas bath for 12-16 hours. <br />
</p><br/><br />
<br />
<p><br />
<font face="Arial, Helvetica"><a name="Cultivate"></a> </font><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Cultivate the Bacteria</b></font></h3><br />
According to the results of the PCR detection, we choose positive colonies and transfer them to 5ml LB liquid media ( 5μl of ampicillin has added) stored in 12.5ml centrifuge tubes. Put the centrifuge tubes in 37℃ gas bath overnight.<br />
</p> <br/><br />
<br />
<p><br />
<font face="Arial, Helvetica"><a name="Flow" ></a> </font><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Flow Cytometer Analysis</b></font></h3><br />
Flow cytometry is a laser based, biophysical technology employed in cell counting, sorting, biomarker detection and protein engineering, by suspending cells in a stream of fluid and passing them by an electronic detection apparatus. It allows simultaneous multiparametric analysis of the physical and/or chemical characteristics of up to thousands of particles per second. <br/><br />
Each fluorophore has a characteristic peak excitation and emission wavelength, and the emission spectra often overlap. Consequently, the combination of labels which can be used depends on the wavelength of the lamp(s) or laser(s) used to excite the fluorochromes and on the detectors available. It is practical that the expression of RFP and GFP can be measured at the same time.<br/><br />
<strong>Method:</strong><br/><br />
1. Materials:<br/><br />
&nbsp;&nbsp;1.1. NaCl solution( 0.9% ,M/V)<br/><br />
&nbsp;&nbsp;1.2. 75% ethanol<br/><br />
2. Procedures:<br/><br />
&nbsp;&nbsp;2.1. Bacterium are harvested by centrifuge at 10000g for 30s, then discard the supernatant. Repeat this step again until we got adequate bacterium.<br/><br />
&nbsp;&nbsp;2.2. Suspend the bacterium With NaCl solution(0.9%) and vibrate the centrifuge tubes until the bacterium are distributed homogeneous.<br/><br />
&nbsp;&nbsp;2.3. Load the bacteria containing pSB1A3 to Flow Cytometer (Beckman), set the parameters. Measure the GFP-FL1 and RFP-FL2 by Cytometer. <br />
</p><br />
<br/><br />
<br />
<p><br />
<font face="Arial, Helvetica"><a name="Fluorescence"></a> </font><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Fluorescence Microscope</b></font></h3><br />
A fluorescence microscope is an optical microscope that uses fluorescence and phosphorescence reflection and absorption to study properties of organic or inorganic substances and generate an image.Fluorescence microscope detection are used to confirm the expression of GFP and RFP which are illuminated with light of a wavelength which excites fluorescence in the sample.<br />
<br/><br />
<strong>Method</strong><br /><br />
Add 10μl of bacteria solution to micro slide and cover with coverslip.<br />
Placed the micro slide on the Fluorescence Microscope. Set parameters to detect GFP and RFP. <br/><br />
Generate an image of fluorescence protein and save it.<br />
</p><br />
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</html></div>M.B.ZHOUhttp://2012.igem.org/Team:SUSTC-Shenzhen-B/protocol1Team:SUSTC-Shenzhen-B/protocol12012-10-26T21:12:43Z<p>M.B.ZHOU: </p>
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.pp_arrow_previous:hover{opacity:.7}div.pp_default .pp_description{font-size:11px;font-weight:700;line-height:14px;margin:5px 50px 5px 0}div.pp_default .pp_bottom .pp_left{background:url(../images/default/sprite.png) -78px -127px no-repeat}div.pp_default .pp_bottom .pp_middle{background:url(../images/default/sprite_x.png) bottom left repeat-x}div.pp_default .pp_bottom .pp_right{background:url(../images/default/sprite.png) -112px -127px no-repeat}div.pp_default .pp_loaderIcon{background:url(../images/default/loader.gif) center center no-repeat}div.light_rounded .pp_top .pp_left{background:url(../images/light_rounded/sprite.png) -88px -53px no-repeat}div.light_rounded .pp_top .pp_right{background:url(../images/light_rounded/sprite.png) -110px -53px no-repeat}div.light_rounded .pp_next:hover{background:url(../images/light_rounded/btnNext.png) center right no-repeat;cursor:pointer}div.light_rounded .pp_previous:hover{background:url(../images/light_rounded/btnPrevious.png) center left no-repeat;cursor:pointer}div.light_rounded .pp_expand{background:url(../images/light_rounded/sprite.png) -31px -26px no-repeat;cursor:pointer}div.light_rounded .pp_expand:hover{background:url(../images/light_rounded/sprite.png) -31px -47px no-repeat;cursor:pointer}div.light_rounded .pp_contract{background:url(../images/light_rounded/sprite.png) 0 -26px no-repeat;cursor:pointer}div.light_rounded .pp_contract:hover{background:url(../images/light_rounded/sprite.png) 0 -47px no-repeat;cursor:pointer}div.light_rounded .pp_close{background:url(../images/light_rounded/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:75px}div.light_rounded .pp_nav .pp_play{background:url(../images/light_rounded/sprite.png) -1px -100px no-repeat;height:15px;width:14px}div.light_rounded .pp_nav .pp_pause{background:url(../images/light_rounded/sprite.png) -24px -100px no-repeat;height:15px;width:14px}div.light_rounded .pp_arrow_previous{background:url(../images/light_rounded/sprite.png) 0 -71px no-repeat}div.light_rounded .pp_arrow_next{background:url(../images/light_rounded/sprite.png) -22px -71px no-repeat}div.light_rounded .pp_bottom .pp_left{background:url(../images/light_rounded/sprite.png) -88px -80px no-repeat}div.light_rounded .pp_bottom .pp_right{background:url(../images/light_rounded/sprite.png) -110px -80px no-repeat}div.dark_rounded .pp_top .pp_left{background:url(../images/dark_rounded/sprite.png) -88px -53px no-repeat}div.dark_rounded .pp_top .pp_right{background:url(../images/dark_rounded/sprite.png) -110px -53px no-repeat}div.dark_rounded .pp_content_container .pp_left{background:url(../images/dark_rounded/contentPattern.png) top left repeat-y}div.dark_rounded .pp_content_container .pp_right{background:url(../images/dark_rounded/contentPattern.png) top right repeat-y}div.dark_rounded .pp_next:hover{background:url(../images/dark_rounded/btnNext.png) center right no-repeat;cursor:pointer}div.dark_rounded .pp_previous:hover{background:url(../images/dark_rounded/btnPrevious.png) center left no-repeat;cursor:pointer}div.dark_rounded .pp_expand{background:url(../images/dark_rounded/sprite.png) -31px -26px no-repeat;cursor:pointer}div.dark_rounded .pp_expand:hover{background:url(../images/dark_rounded/sprite.png) -31px -47px no-repeat;cursor:pointer}div.dark_rounded .pp_contract{background:url(../images/dark_rounded/sprite.png) 0 -26px no-repeat;cursor:pointer}div.dark_rounded .pp_contract:hover{background:url(../images/dark_rounded/sprite.png) 0 -47px no-repeat;cursor:pointer}div.dark_rounded .pp_close{background:url(../images/dark_rounded/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:75px}div.dark_rounded .pp_description{color:#fff;margin-right:85px}div.dark_rounded .pp_nav .pp_play{background:url(../images/dark_rounded/sprite.png) -1px -100px no-repeat;height:15px;width:14px}div.dark_rounded .pp_nav .pp_pause{background:url(../images/dark_rounded/sprite.png) -24px -100px no-repeat;height:15px;width:14px}div.dark_rounded .pp_arrow_previous{background:url(../images/dark_rounded/sprite.png) 0 -71px no-repeat}div.dark_rounded .pp_arrow_next{background:url(../images/dark_rounded/sprite.png) -22px -71px no-repeat}div.dark_rounded .pp_bottom .pp_left{background:url(../images/dark_rounded/sprite.png) -88px -80px no-repeat}div.dark_rounded .pp_bottom .pp_right{background:url(../images/dark_rounded/sprite.png) -110px -80px no-repeat}div.dark_rounded .pp_loaderIcon{background:url(../images/dark_rounded/loader.gif) center center no-repeat}div.dark_square .pp_left,div.dark_square .pp_middle,div.dark_square .pp_right,div.dark_square .pp_content{background:#000}div.dark_square .pp_description{color:#fff;margin:0 85px 0 0}div.dark_square .pp_loaderIcon{background:url(../images/dark_square/loader.gif) center center no-repeat}div.dark_square .pp_expand{background:url(../images/dark_square/sprite.png) -31px -26px no-repeat;cursor:pointer}div.dark_square .pp_expand:hover{background:url(../images/dark_square/sprite.png) -31px -47px no-repeat;cursor:pointer}div.dark_square .pp_contract{background:url(../images/dark_square/sprite.png) 0 -26px no-repeat;cursor:pointer}div.dark_square .pp_contract:hover{background:url(../images/dark_square/sprite.png) 0 -47px no-repeat;cursor:pointer}div.dark_square .pp_close{background:url(../images/dark_square/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:75px}div.dark_square .pp_nav{clear:none}div.dark_square .pp_nav .pp_play{background:url(../images/dark_square/sprite.png) -1px -100px no-repeat;height:15px;width:14px}div.dark_square .pp_nav .pp_pause{background:url(../images/dark_square/sprite.png) -24px -100px no-repeat;height:15px;width:14px}div.dark_square .pp_arrow_previous{background:url(../images/dark_square/sprite.png) 0 -71px no-repeat}div.dark_square .pp_arrow_next{background:url(../images/dark_square/sprite.png) -22px -71px no-repeat}div.dark_square .pp_next:hover{background:url(../images/dark_square/btnNext.png) center right no-repeat;cursor:pointer}div.dark_square .pp_previous:hover{background:url(../images/dark_square/btnPrevious.png) center left no-repeat;cursor:pointer}div.light_square .pp_expand{background:url(../images/light_square/sprite.png) -31px -26px no-repeat;cursor:pointer}div.light_square .pp_expand:hover{background:url(../images/light_square/sprite.png) -31px -47px no-repeat;cursor:pointer}div.light_square .pp_contract{background:url(../images/light_square/sprite.png) 0 -26px no-repeat;cursor:pointer}div.light_square .pp_contract:hover{background:url(../images/light_square/sprite.png) 0 -47px no-repeat;cursor:pointer}div.light_square .pp_close{background:url(../images/light_square/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:75px}div.light_square .pp_nav .pp_play{background:url(../images/light_square/sprite.png) -1px -100px no-repeat;height:15px;width:14px}div.light_square .pp_nav .pp_pause{background:url(../images/light_square/sprite.png) -24px -100px no-repeat;height:15px;width:14px}div.light_square .pp_arrow_previous{background:url(../images/light_square/sprite.png) 0 -71px no-repeat}div.light_square .pp_arrow_next{background:url(../images/light_square/sprite.png) -22px -71px no-repeat}div.light_square .pp_next:hover{background:url(../images/light_square/btnNext.png) center right no-repeat;cursor:pointer}div.light_square .pp_previous:hover{background:url(../images/light_square/btnPrevious.png) center left no-repeat;cursor:pointer}div.facebook .pp_top .pp_left{background:url(../images/facebook/sprite.png) -88px -53px no-repeat}div.facebook .pp_top .pp_middle{background:url(../images/facebook/contentPatternTop.png) top left repeat-x}div.facebook .pp_top .pp_right{background:url(../images/facebook/sprite.png) -110px -53px no-repeat}div.facebook .pp_content_container .pp_left{background:url(../images/facebook/contentPatternLeft.png) top left repeat-y}div.facebook .pp_content_container .pp_right{background:url(../images/facebook/contentPatternRight.png) top right repeat-y}div.facebook .pp_expand{background:url(../images/facebook/sprite.png) -31px -26px no-repeat;cursor:pointer}div.facebook .pp_expand:hover{background:url(../images/facebook/sprite.png) -31px -47px no-repeat;cursor:pointer}div.facebook .pp_contract{background:url(../images/facebook/sprite.png) 0 -26px no-repeat;cursor:pointer}div.facebook .pp_contract:hover{background:url(../images/facebook/sprite.png) 0 -47px no-repeat;cursor:pointer}div.facebook .pp_close{background:url(../images/facebook/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:22px}div.facebook .pp_description{margin:0 37px 0 0}div.facebook .pp_loaderIcon{background:url(../images/facebook/loader.gif) center center no-repeat}div.facebook .pp_arrow_previous{background:url(../images/facebook/sprite.png) 0 -71px no-repeat;height:22px;margin-top:0;width:22px}div.facebook .pp_arrow_previous.disabled{background-position:0 -96px;cursor:default}div.facebook .pp_arrow_next{background:url(../images/facebook/sprite.png) -32px -71px no-repeat;height:22px;margin-top:0;width:22px}div.facebook .pp_arrow_next.disabled{background-position:-32px -96px;cursor:default}div.facebook .pp_nav{margin-top:0}div.facebook .pp_nav p{font-size:15px;padding:0 3px 0 4px}div.facebook .pp_nav .pp_play{background:url(../images/facebook/sprite.png) -1px -123px no-repeat;height:22px;width:22px}div.facebook .pp_nav .pp_pause{background:url(../images/facebook/sprite.png) -32px -123px no-repeat;height:22px;width:22px}div.facebook .pp_next:hover{background:url(../images/facebook/btnNext.png) center right no-repeat;cursor:pointer}div.facebook .pp_previous:hover{background:url(../images/facebook/btnPrevious.png) center left no-repeat;cursor:pointer}div.facebook .pp_bottom .pp_left{background:url(../images/facebook/sprite.png) -88px -80px no-repeat}div.facebook .pp_bottom .pp_middle{background:url(../images/facebook/contentPatternBottom.png) top left repeat-x}div.facebook .pp_bottom .pp_right{background:url(../images/facebook/sprite.png) -110px -80px no-repeat}div.pp_pic_holder a:focus{outline:0}div.pp_overlay{background:#000;display:none;left:0;position:absolute;top:0;width:100%;z-index:9500}div.pp_pic_holder{display:none;position:absolute;width:100px;z-index:10000}.pp_content{height:40px;min-width:40px}* html .pp_content{width:40px}.pp_content_container{position:relative;text-align:left;width:100%}.pp_content_container .pp_left{padding-left:20px}.pp_content_container .pp_right{padding-right:20px}.pp_content_container .pp_details{float:left;margin:10px 0 2px}.pp_description{display:none;margin:0}.pp_social{float:left;margin:0}.pp_social .facebook{float:left;margin-left:5px;overflow:hidden;width:55px}.pp_social .twitter{float:left}.pp_nav{clear:right;float:left;margin:3px 10px 0 0}.pp_nav p{float:left;margin:2px 4px;white-space:nowrap}.pp_nav .pp_play,.pp_nav .pp_pause{float:left;margin-right:4px;text-indent:-10000px}a.pp_arrow_previous,a.pp_arrow_next{display:block;float:left;height:15px;margin-top:3px;overflow:hidden;text-indent:-10000px;width:14px}.pp_hoverContainer{position:absolute;top:0;width:100%;z-index:2000}.pp_gallery{display:none;left:50%;margin-top:-50px;position:absolute;z-index:10000}.pp_gallery div{float:left;overflow:hidden;position:relative}.pp_gallery ul{float:left;height:35px;margin:0 0 0 5px;padding:0;position:relative;white-space:nowrap}.pp_gallery ul a{border:1px rgba(0,0,0,.5) solid;display:block;float:left;height:33px;overflow:hidden}.pp_gallery ul a img{border:0}.pp_gallery li{display:block;float:left;margin:0 5px 0 0;padding:0}.pp_gallery li.default a{background:url(../images/facebook/default_thumbnail.gif) 0 0 no-repeat;display:block;height:33px;width:50px}.pp_gallery .pp_arrow_previous,.pp_gallery .pp_arrow_next{margin-top:7px!important}a.pp_next{background:url(../images/light_rounded/btnNext.png) 10000px 10000px no-repeat;display:block;float:right;height:100%;text-indent:-10000px;width:49%}a.pp_previous{background:url(../images/light_rounded/btnNext.png) 10000px 10000px no-repeat;display:block;float:left;height:100%;text-indent:-10000px;width:49%}a.pp_expand,a.pp_contract{cursor:pointer;display:none;height:20px;position:absolute;right:30px;text-indent:-10000px;top:10px;width:20px;z-index:20000}a.pp_close{display:block;line-height:22px;position:absolute;right:0;text-indent:-10000px;top:0}.pp_loaderIcon{display:block;height:24px;left:50%;margin:-12px 0 0 -12px;position:absolute;top:50%;width:24px}#pp_full_res{line-height:1!important}#pp_full_res .pp_inline{text-align:left}#pp_full_res .pp_inline p{margin:0 0 15px}div.ppt{color:#fff;display:none;font-size:17px;margin:0 0 5px 15px;z-index:9999}div.pp_default .pp_content,div.light_rounded .pp_content{background-color:#fff}div.pp_default #pp_full_res .pp_inline,div.light_rounded .pp_content .ppt,div.light_rounded #pp_full_res .pp_inline,div.light_square .pp_content .ppt,div.light_square #pp_full_res .pp_inline,div.facebook .pp_content .ppt,div.facebook #pp_full_res .pp_inline{color:#000}div.pp_default .pp_gallery ul li a:hover,div.pp_default .pp_gallery ul li.selected a,.pp_gallery ul a:hover,.pp_gallery li.selected a{border-color:#fff}div.pp_default .pp_details,div.light_rounded .pp_details,div.dark_rounded .pp_details,div.dark_square .pp_details,div.light_square .pp_details,div.facebook .pp_details{position:relative}div.light_rounded .pp_top .pp_middle,div.light_rounded .pp_content_container .pp_left,div.light_rounded .pp_content_container .pp_right,div.light_rounded .pp_bottom .pp_middle,div.light_square .pp_left,div.light_square .pp_middle,div.light_square .pp_right,div.light_square .pp_content,div.facebook .pp_content{background:#fff}div.light_rounded .pp_description,div.light_square .pp_description{margin-right:85px}div.light_rounded .pp_gallery a.pp_arrow_previous,div.light_rounded .pp_gallery a.pp_arrow_next,div.dark_rounded .pp_gallery a.pp_arrow_previous,div.dark_rounded .pp_gallery a.pp_arrow_next,div.dark_square .pp_gallery a.pp_arrow_previous,div.dark_square .pp_gallery a.pp_arrow_next,div.light_square .pp_gallery a.pp_arrow_previous,div.light_square .pp_gallery a.pp_arrow_next{margin-top:12px!important}div.light_rounded .pp_arrow_previous.disabled,div.dark_rounded .pp_arrow_previous.disabled,div.dark_square .pp_arrow_previous.disabled,div.light_square .pp_arrow_previous.disabled{background-position:0 -87px;cursor:default}div.light_rounded .pp_arrow_next.disabled,div.dark_rounded .pp_arrow_next.disabled,div.dark_square .pp_arrow_next.disabled,div.light_square .pp_arrow_next.disabled{background-position:-22px -87px;cursor:default}div.light_rounded .pp_loaderIcon,div.light_square .pp_loaderIcon{background:url(../images/light_rounded/loader.gif) center center no-repeat}div.dark_rounded .pp_top .pp_middle,div.dark_rounded .pp_content,div.dark_rounded .pp_bottom .pp_middle{background:url(../images/dark_rounded/contentPattern.png) top left repeat}div.dark_rounded .currentTextHolder,div.dark_square .currentTextHolder{color:#c4c4c4}div.dark_rounded #pp_full_res .pp_inline,div.dark_square #pp_full_res .pp_inline{color:#fff}.pp_top,.pp_bottom{height:20px;position:relative}* html .pp_top,* html .pp_bottom{padding:0 20px}.pp_top .pp_left,.pp_bottom .pp_left{height:20px;left:0;position:absolute;width:20px}.pp_top .pp_middle,.pp_bottom .pp_middle{height:20px;left:20px;position:absolute;right:20px}* html .pp_top .pp_middle,* html .pp_bottom .pp_middle{left:0;position:static}.pp_top .pp_right,.pp_bottom .pp_right{height:20px;left:auto;position:absolute;right:0;top:0;width:20px}.pp_fade,.pp_gallery li.default a img{display:none}<br />
<br />
</style><br />
<script type="text/javascript"><br />
<br />
/*<br />
* Superfish v1.4.8 - jQuery menu widget<br />
* Copyright (c) 2008 Joel Birch<br />
*<br />
* Dual licensed under the MIT and GPL licenses:<br />
* http://www.opensource.org/licenses/mit-license.php<br />
* http://www.gnu.org/licenses/gpl.html<br />
*<br />
* CHANGELOG: http://users.tpg.com.au/j_birch/plugins/superfish/changelog.txt<br />
*/<br />
<br />
;(function($){<br />
$.fn.superfish = function(op){<br />
<br />
var sf = $.fn.superfish,<br />
c = sf.c,<br />
$arrow = $(['<span class="',c.arrowClass,'"> &#187;</span>'].join('')),<br />
over = function(){<br />
var $$ = $(this), menu = getMenu($$);<br />
clearTimeout(menu.sfTimer);<br />
$$.showSuperfishUl().siblings().hideSuperfishUl();<br />
},<br />
out = function(){<br />
var $$ = $(this), menu = getMenu($$), o = sf.op;<br />
clearTimeout(menu.sfTimer);<br />
menu.sfTimer=setTimeout(function(){<br />
o.retainPath=($.inArray($$[0],o.$path)>-1);<br />
$$.hideSuperfishUl();<br />
//if (o.$path.length &amp;&amp; $$.parents(['li.',o.hoverClass].join('')).length<1){over.call(o.$path);}<br />
if (o.$path.length) {<br />
if ($$.parents(['li.',o.hoverClass].join('')).length<1){over.call(o.$path);}<br />
}<br />
},o.delay); <br />
},<br />
getMenu = function($menu){<br />
var menu = $menu.parents(['ul.',c.menuClass,':first'].join(''))[0];<br />
sf.op = sf.o[menu.serial];<br />
return menu;<br />
},<br />
addArrow = function($a){ $a.addClass(c.anchorClass).append($arrow.clone()); };<br />
<br />
return this.each(function() {<br />
var s = this.serial = sf.o.length;<br />
var o = $.extend({},sf.defaults,op);<br />
o.$path = $('li.'+o.pathClass,this).slice(0,o.pathLevels).each(function(){<br />
$(this).addClass([o.hoverClass,c.bcClass].join(' '))<br />
.filter('li:has(ul)').removeClass(o.pathClass);<br />
});<br />
sf.o[s] = sf.op = o;<br />
var bcheck = false;<br />
if ($.fn.hoverIntent) {<br />
if (!o.disableHI) bcheck = true;<br />
}<br />
<br />
$('li:has(ul)',this)[(bcheck) ? 'hoverIntent' : 'hover'](over,out).each(function() {<br />
if (o.autoArrows) addArrow( $('>a:first-child',this) );<br />
})<br />
.not('.'+c.bcClass)<br />
.hideSuperfishUl();<br />
<br />
var $a = $('a',this);<br />
$a.each(function(i){<br />
var $li = $a.eq(i).parents('li');<br />
$a.eq(i).focus(function(){over.call($li);}).blur(function(){out.call($li);});<br />
});<br />
o.onInit.call(this);<br />
<br />
}).each(function() {<br />
var menuClasses = [c.menuClass];<br />
//if (sf.op.dropShadows &amp;&amp; !($.browser.msie &amp;&amp; $.browser.version < 7)) menuClasses.push(c.shadowClass);<br />
if (sf.op.dropShadows) if (!$.browser.msie) if (!($.browser.version < 7)) menuClasses.push(c.shadowClass);<br />
$(this).addClass(menuClasses.join(' '));<br />
});<br />
};<br />
<br />
var sf = $.fn.superfish;<br />
sf.o = [];<br />
sf.op = {};<br />
sf.IE7fix = function(){<br />
var o = sf.op;<br />
//if ($.browser.msie &amp;&amp; $.browser.version > 6 &amp;&amp; o.dropShadows &amp;&amp; o.animation.opacity!=undefined)<br />
if ($.browser.msie) if($.browser.version > 6) if (o.dropShadows) if (o.animation.opacity!=undefined)<br />
this.toggleClass(sf.c.shadowClass+'-off');<br />
};<br />
sf.c = {<br />
bcClass : 'sf-breadcrumb',<br />
menuClass : 'sf-js-enabled',<br />
anchorClass : 'sf-with-ul',<br />
arrowClass : 'sf-sub-indicator',<br />
shadowClass : 'sf-shadow'<br />
};<br />
sf.defaults = {<br />
hoverClass : 'sfHover',<br />
pathClass : 'overideThisToUse',<br />
pathLevels : 1,<br />
delay : 800,<br />
animation : {opacity:'show'},<br />
speed : 'normal',<br />
autoArrows : true,<br />
dropShadows : true,<br />
disableHI : false, // true disables hoverIntent detection<br />
onInit : function(){}, // callback functions<br />
onBeforeShow: function(){},<br />
onShow : function(){},<br />
onHide : function(){}<br />
};<br />
$.fn.extend({<br />
hideSuperfishUl : function(){<br />
var o = sf.op,<br />
not = (o.retainPath===true) ? o.$path : '';<br />
o.retainPath = false;<br />
var $ul = $(['li.',o.hoverClass].join(''),this).add(this).not(not).removeClass(o.hoverClass)<br />
.find('>ul').hide().css('visibility','hidden');<br />
o.onHide.call($ul);<br />
return this;<br />
},<br />
showSuperfishUl : function(){<br />
var o = sf.op,<br />
sh = sf.c.shadowClass+'-off',<br />
$ul = this.addClass(o.hoverClass)<br />
.find('>ul:hidden').css('visibility','visible');<br />
sf.IE7fix.call($ul);<br />
o.onBeforeShow.call($ul);<br />
$ul.animate(o.animation,o.speed,function(){ sf.IE7fix.call($ul); o.onShow.call($ul); });<br />
return this;<br />
}<br />
});<br />
<br />
})(jQuery);<br />
<br />
/*<br />
* Supersubs v0.2b - jQuery plugin<br />
* Copyright (c) 2008 Joel Birch<br />
*<br />
* Dual licensed under the MIT and GPL licenses:<br />
* http://www.opensource.org/licenses/mit-license.php<br />
* http://www.gnu.org/licenses/gpl.html<br />
*<br />
*<br />
* This plugin automatically adjusts submenu widths of suckerfish-style menus to that of<br />
* their longest list item children. If you use this, please expect bugs and report them<br />
* to the jQuery Google Group with the word 'Superfish' in the subject line.<br />
*<br />
*/<br />
<br />
(function($){ // $ will refer to jQuery within this closure<br />
<br />
$.fn.supersubs = function(options){<br />
var opts = $.extend({}, $.fn.supersubs.defaults, options);<br />
// return original object to support chaining<br />
return this.each(function() {<br />
// cache selections<br />
var $$ = $(this);<br />
// support metadata<br />
var o = $.meta ? $.extend({}, opts, $$.data()) : opts;<br />
// get the font size of menu.<br />
// .css('fontSize') returns various results cross-browser, so measure an em dash instead<br />
var fontsize = $('<li id="menu-fontsize">&#8212;</li>').css({<br />
'padding' : 0,<br />
'position' : 'absolute',<br />
'top' : '-999em',<br />
'width' : 'auto'<br />
}).appendTo($$).width(); //clientWidth is faster, but was incorrect here<br />
// remove em dash<br />
$('#menu-fontsize').remove();<br />
// cache all ul elements<br />
$ULs = $$.find('ul');<br />
// loop through each ul in menu<br />
$ULs.each(function(i) { <br />
// cache this ul<br />
var $ul = $ULs.eq(i);<br />
// get all (li) children of this ul<br />
var $LIs = $ul.children();<br />
// get all anchor grand-children<br />
var $As = $LIs.children('a');<br />
// force content to one line and save current float property<br />
var liFloat = $LIs.css('white-space','nowrap').css('float');<br />
// remove width restrictions and floats so elements remain vertically stacked<br />
var emWidth = $ul.add($LIs).add($As).css({<br />
'float' : 'none',<br />
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<article><br />
<h1><p>Lab Protocol</p></h1><br />
</font><br />
<font face="Arial, Helvetica"><br />
<p><a href="#Site-Directed_Mutagenesis">1. Site-Directed Mutagenesis</a></p><br />
<p><a href="#Restriction">2. Mutation Verification by Restriction Enzyme Digestion</a></p><br />
<p><a href="#Media">3. Media Preparation</a></p><br />
<p><a href="#Bacterial">4. Bacterial Transformation</a></p><br />
<p><a href="#Colony">5. Colony PCR for Verification</a></p><br />
<p><a href="#Culture">6. Cultivate the Bacteria</a></p><br />
<p><a href="#Plasmid">7. Plasmid DNA Isolation</a></p><br />
<p><a href="#Restriction_2">8. Mutation Verification by Restriction Enzyme Digestion </a></p><br />
<p><a href="#Amplify">9. Polymerase Chain Reaction and Electrophoresis</a></p><br />
<p><a href="#Electrophoresis">10. Double Restriction Enzyme Digestion and Electrophoresis </a></p><br />
<p><a href="#Ligation">11. Ligation</a></p><br />
<p><a href="#Bacterial_Transformation">12. Bacterial Transformation</a></p><br />
<p><a href="#bacterial_colony">13. Bacterial Colony PCR</a></p><br />
<p><a href="#Culture_the">14. Cultivate the Bacteria</a></p><br />
<p><a href="#Plasmid_DNA">15. Plasmid DNA Isolation</a></p><br />
<p><a href="#Restriction_Enzyme">16. Restriction Enzyme Digestion and Electrophoresis</a></p><br />
<p><a href="#Ligation1">17. Ligation</a></p><br />
<p><a href="#Bacteria">18. Bacteria Transformation</a></p><br />
<p><a href="#Cultivate">19. Cultivate the Bacteria</a></p><br />
<p><a href="#Flow">20. Flow Cytometer Analysis</a></p><br />
<p><a href="#Fluorescence">21. Fluorescence Microscope </a></p><br />
<br/><br />
<br />
<p><br />
<a name="Site-Directed_Mutagenesis" ></a></font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Site-Directed Mutagenesis</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
Plasmid pSB1A3 was chosen as a backbone vector for cloning. The Pst I site in pSB1A3 was mutated to Afl II site to facilitate following cloning processes. Proper primers were designed and PCR-based site-directed mutageneis were carried out to generate this mutation as descbibed below. <br/><br />
<br />
<!--<p>要到很后面才能找到他的结束--><br />
<strong>Method:</strong><br/><br />
<br />
1. Set up PCR assay tubes as described below:<br />
<br/><br />
Total: 25 μl<br /><br />
+ 0.25 μl of Ex Taq polymerase <br /><br />
+ 2.5 μl of 10× Taq reaction buffer<br /><br />
+ 2.0 μl of dNTP(2 mM) <br /><br />
+ 1.0 μl of template (E.coli plasmid 817) <br /><br />
+ 1.0 μl of oligonucleotide primer PtoA-F*<br /><br />
+ 1.0 μl of oligonucleotide primer PtoA-R* <br /><br />
+ 18.25 μl of ddH2O <br /><br />
*The sequences of primer pair PtoA-F and PtoA-R.<br /><br />
PtoA-F 5'-CCACCTGACGTCTAAGAAAC-3'<br /><br />
PtoA-R 5'-ATGATCATCGCCGGCGAATTCAGGC-3' <br /><br />
<br />
2. Set parameters for PCR to amplify desired products. <br/><br />
<br />
<table><br />
<tr><br />
<td>Temperature</td> <td>Time</td> <td>Cycle</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>5min</td> <td>1</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>55˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>72˚C</td> <td>1min 20sec</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>4˚C</td> <td>7hrs</td> <td>1</td><br />
</tr><br />
</table><br />
<!--那个p的结束,下同--><br />
</p><br />
<br />
<p><br />
<font face="Arial, Helvetica"><br />
<br />
<a name="Restriction" ></a> <br />
</font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Restriction Enzyme Digestion for Verification</font></b></font></h3><br />
To verify whether PstI site in pSB1A3 was successfully mutated to AflII site, we performed restriction enzyme digestion experiments. <br/><br />
<br />
Bacterial plasmids are double-stranded circular DNA molecules and uncut plasmid DNA can be in any of three forms - nicked circular, linear, closed supercoiled. When run on an agarose gel one frequently will see these forms as different bands with closed supercoiled form migrates the fastest, linear form migrates the slowest, and nicked circular migrates in between. If the PStI site was successfully mutated to AflII site, we expected to see increased linear form of pSB1A3 when digested with AflII. SpeI-cut pSB1A3 was served as a positive control. <br /><br />
<br />
<strong>Method</strong><br /><br />
1. Set up Pst I digestion in 1.5 ml eppendorf tubes as described below:<br /><br />
Total: 10μl<br /><br />
+ 0.5μl of Pst I restriction enzyme (company :Takara)<br /><br />
+ 1μl of 10XH buffer<br /><br />
+ 1μl of plasmid DNA<br /><br />
+ 7.5μl of ddH2O <br /><br />
2. Set up Afl II digestion in 1.5 ml eppendorf tubes as described below:<br /><br />
Total: 10μl<br /><br />
+ 0.5μl of Afl II restriction enzyme, (company :Takara)<br /><br />
+ 1μl of 10XM buffer<br /><br />
+ 1μl of plasmid DNA<br /><br />
+ 1.0μl of 0.01% BSA<br /><br />
+ 6.5μl of ddH2O<br /><br />
3. Incubate the eppendorf tubes in 37℃ water bath for 1-2 hr. <br/><br />
4. Prepare 1% agarose gel in Conical flask. Weigh 0.6 g agarose and add 60 ml 1x TAE (diluted from 50x TAE). Cover the Conical flask with silver paper to avoid the loss of water vapor. Place the Conical flask in the microwave and microwave for 1 minute with a middle power. Take it out and shake gently till the solution is homogeneous,(BE CAREFUL to watch the solution closely when shake it–it superheats and can boil over and cause severe burns). Continue microwave and swirl until solution is seen clear and homogeneous with no existence of solid. After cool down the agarose gel briefly, add 3 μl of Gelred (10000x ) and mix well. Pour the agarose gel in gel casting apparatus and insert combs.<br/><br />
5. By inserting the pipette tip below the TAE liquid and into the well, add 5 μl of 1kb DNA ladder solution to first (and last if desired) well, skip one well, then begin adding the 5μl of digested DNA solutions mixed with 1 μl loading buffer (6x) to the wells. <br/><br />
6. Place the cover on the electrophoresis unit, plug into the power source, and turn on voltage to 120V, set time to 30 minutes, and press the start button twice,until the bubbles are seen. DNA separation can be observed as time goes on by turning off the power supply then gently removing the basin from the electrophoresis unit (be careful not to let the gel slip out of the basin) and placing on the UV transilluminator to see DNA bands. <br/><br />
7. When the desired level of separation is obtained, the basin can be placed on the transilluminator for picture taking(Of the absence of transilluminator,we use camera to take pictures with the UV light ). <br/><br />
8. Cut the gel of specific position and collect it in tubes that have measured weight. <br/><br />
9. Use DNA Gel Extraction Ki to purify the plasmid DNA mutant-pSB1A3.<br />
RPF (SpeI/AflII-digested), GFP (SpeI/AflII-digested) fragments were ligated into this vector, which was followed by insertion of designed terminator sequences between RFP and GFP, respectively.</p><br />
<font face="Arial, Helvetica"><br />
<img src="https://static.igem.org/mediawiki/2012/5/59/11.png" alt="" class="img_fl img_border" align="left"/> <br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
(Figure 1 : This figure shows that the site-directed mutagenesis succeed ,we successfully change a restriction enzyme cutting site named Pst I to Afl II. Lane 1 represents the plasmid mutant-pSB1A3, lane 2 shows that the mutant-pSB1A3 cannot be digested by restriction enzyme Spe I ,lane 3 shows that mutant-pSB1A3 can be digested by restriction enzyme Afl II.) <br />
</p><br />
<br />
<p><br />
<a name="Media"></a></font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Media Preparation</font></b></font></h3><br />
For all experiments involving the bacterial biomass and experimentation, proper media is chosen to grow the cells. Commonly,we use Lysogeny broth media for <em>E. coli</em>. The following is the media compositions and their quantities.<br /><br />
<strong>Method:</strong><br/><br />
<br />
1.Prepare the Lysogeny Broth (LB) liquid media (1 L) as indicated below: <br/><br />
+ Bacto-Tryptone - 10 g<br/><br />
+ NaCl - 10 g<br/><br />
+ Yeast Extract - 5 g<br/><br />
Add ddH2O and 5mmol/L Tris Buffer in a measuring cylinder to ensure accuracy, to make a total of 1 liter and pH is 8.0.<br/><br />
2.Prepare the Lysogeny Broth (LB) solid media (1 L) as indicated below:<br/><br />
+ Bacto-Tryptone - 10 g<br/><br />
+ NaCl - 10 g<br/><br />
+ Yeast Extract - 5 g<br/><br />
+ Difco Agar - 15g<br/><br />
Add ddH2O and 5mmol/L Tris Buffer in a measuring cylinder to ensure accuracy, to make a total of 1 liter and pH is 8.0.<br/><br />
3. Autoclaving<br/><br />
Autoclave at 121 °C for 60 minutes. After the media cooling down enough, antibiotics Ampicillin(100mg of Ampicillin per 1ml of the media) are added. At last the media are poured 15ml on each plate and become solid.Store the plate at 4℃ refrigerator.<br />
</p><br />
<br />
<p><br />
<font face="Arial, Helvetica"><br />
<br><br />
<a name="Bacterial"></a></font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Bacterial Transformation</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
Transformation is commonly used to introduce recombinant plasmid DNA into bacterial strains which can transform naturally or can be made competitive for transformation by artificial means. The purpose of this technique is to introduce a recombinant plasmid DNA into a bacterial strains and to use bacteria strains to amplify the plasmid mutant-pSB1A3 for further plasmid construction.<br />
<br />
<strong>Method</strong><br /><br />
1. Take out an appropriate number of tubes that contain competent cells(100μl ) from the freezer. Immediately place the tubes on ice, so that all but the cap is surrounded by ice. Allow the cells to thaw on ice for 2-5 min.<br /><br />
2. Visually check the cells to see whether they have thawed and gently flick the cells 1-2 times to evenly resuspend the cells.<br /><br />
3. Add 10μl PCR products(mini-prep purified) to the competent cells DH-5α. Stir gently to mix and return the tube to the ice, making sure that the tube is surrounded by ice except for the cap. Repeat for additional two times for the same samples.<br /><br />
4. Incubate the tubes on ice for 30 min.<br /><br />
5. Place the tubes in a 42°C water bath for exactly 90 sec; do not shake.<br /><br />
6. Place the tubes on ice for 2 min to cool down.<br /><br />
7. Add 800 l of room temperature LB medium to each tube.<br /><br />
8. Shake the tubes vigorously at 37<a name="OLE_LINK2" id="OLE_LINK2"></a><a name="OLE_LINK1" id="OLE_LINK1">°C</a> for 45-60 min.<br /><br />
9. Centrifuge the tubes at 3K RPM for 1 min. Discard the supernatant liquor and leave 100-200 μl of the mixtures.Mix the contents and spread the whole liquid on LB agar plates containing the appropriate antibiotic ampicillin for the plasmid.<br /><br />
10. Place the plates on the bench for several min to allow excess liquid to be absorbed, and then invert and incubate overnight at 37°C (12-16 h).</p><br />
<br/><br />
</p><br />
<br />
<p><br />
<a name="Colony"></a></font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Colony PCR for Verification</font> </b></font></h3><br />
<font face="Arial, Helvetica"><br />
Colony PCR is used to identify and select cell colonies that have the correct plasmid inserted. The procedure is a way to do several PCR operations on cell colonies in parallel, to evaluate the results and select the corresponding positive cell colonies. After an overnight growth of E.coli, we can pick up several colonies from the plate and do a colony PCR verification. Besides, the colonies we choose and should also be stored, we can incubate these colonies in one plate after every colony has been marked.<br />
<br />
<strong>Method</strong><br /><br />
1. Prepare the sample reaction as indicated below:<br /><br />
Total: 25μl <br /><br />
+ 0.25 μl of Ex Taq polymerase (company:Takara)<br /><br />
+ 2.5 μl of 10× Taq reaction buffer<br /><br />
+ 1.0 μl of R-NPS-F*<br /><br />
+ 1.0 μl of G-SXA-R*<br /><br />
+ 1.0 μl of plasmid mutant-pSB1A3<br /><br />
+ 2.0 μl of dNTP( 25mM ) <br /><br />
+ 17.25 μl of ddH2O <br /><br />
Note: The sequences of primers R-NPS-F , G-SXA-R <br /><br />
R-NPS-F 5'-TATAGCGGCCGCCTTAAGTAAGTAAGAGTATACG-3' <br /><br />
G-SXA-R 5'-TCTCCGACTTAAGGGATCCTATAAACGCAG-3'<br /><br />
<br />
2. Set parameters for PCR to amplify desired products. <br />
<br />
<table><br />
<tr><br />
<td>Temperature</td> <td>Time</td> <td>Cycle</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>5min</td> <td>1</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>55˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>72˚C</td> <td>1min 20sec</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>4˚C</td> <td>7hrs</td> <td>1</td><br />
</tr><br />
</table><br />
<br />
3. Electrophorese the total system and observe the lane separation. <br />
<br><br />
<a name="Culture"></a></font><br />
<br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Cultivate the Bacteria</b></font></h3><br />
<font face="Arial, Helvetica"><p>According to the results of the PCR detection, positive colonies are chosen and transferred them to 5ml LB liquid media ( 5μl of ampicillin added) stored in 12.5ml centrifuge tubes. Put the centrifuge tubes in 37℃ gas bath overnight. <br />
<br><br />
<a name="Plasmid"></a><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Plasmid DNA Isolation</b></font></h3><br />
Use E.Z.N.A.TM Plasmid Mini I to realize plasmid DNA isolation.<br /><br />
<strong>Method</strong><br/><br />
<br />
<ol><br />
<li>Transfer 5 ml of overnight culture into a 1.5-ml eppendorf tube labeled with group number. </li><br />
<li>Centrifuge the sample at max. speed of desk top centrifuge and RT for 1min to pellet the cells.</li><br />
<li>Discard the supernatant. Remove as much of the supernatant as possible without disturbing the cell pellet. </li><br />
<li>Repeat step 1 and 2 twice. </li><br />
<li>Resuspend the pellet completely in 250 ml of Solution I (containing RNase A) by vortexing the samples vigorously . No clumps should be visible in the tube. </li><br />
<li>Add 250 ml of Solution II and mix the sample by gently inverting the tube 4 to 6 times. Do not vortex or shake the sample vigorously. The bacterial suspension should begin to clear which have lysed the bacterial cells in this step. <strong>Warning: </strong>Do not stop here for more than five min, as the high pH hurts your DNA! </li><br />
<li>Add 350 ml of Solution III and mix by gently inverting the tube 4 to 6 times until a flocculent white precipitate forms. Do not shake vigorously, as it might break the genomic DNA. </li><br />
<li>Centrifuge at maximum speed for 10 min at room temperature to pellet the cell debris. You should see a white precipitate in the tube after the centrifugation. </li><br />
<li>While the samples are centrifuging, for each sample, label a clean HiBind Miniprep Column which is to assembled in a 2-ml collection tube</li><br />
<li>Apply the supernatants from step 8 to the columns. </li><br />
<li>Centrifuge at maximum speed for 1 min at RT. Discard the flow-through in the collection tube. </li><br />
<li>Add 500 ml of Buffer HB to wash the Hibind Miniprep Column. Centrifuge at maximum speed for 1 min at RT. Discard the flow-through in the collection tube. </li><br />
<li>Wash the column by adding 700 ml of DNA Wash Buffer diluted with absolute ethanol. Centrifuge at maximum speed for 1 min at room temperature and discard the flow-through.</li><br />
<li>Then centrifuge the tubes again for 2 min to remove all the moisture.</li><br />
<li>Place the column in a clean 1.5 ml eppendorf tube that is labeled with the plasmid name and group number. To elute the DNA, add 50 ml of Elution Buffer to the center of each column. Let the samples stand for 2 or more minutes at RT, and then centrifuge for 1 min. The sample in the centrifuge tube (bottom) is your plasmid DNA. </li><br />
<li>Discard the column and save the sample in the eppendorf tube by placing it in the freezer (-20°C). </li><br />
</ol><br />
</p><br />
<br />
<br />
<a name="#Restriction_2" ></a> <br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Restriction Enzyme Digestion and Electrophoresis</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Because the colony PCR test is so sensitive and affect markedly by environment factors. So we do a restriction enzyme digestion to ensure that the isolated plasmid is the site-directed mutated plasmid.<br /><br />
<strong>Method</strong><br /><br />
1. Prepare the control reaction as indicated below:<br /><br />
Total: 10μl<br /><br />
+ 0.5μl of Pst I restriction enzyme(company :Takara)<br /><br />
+ 1μl of 10XH buffer<br /><br />
+ 1μl of plasmid DNA<br /><br />
+ 7.5μl of ddH2O <br /><br />
2. Prepare the sample reaction as indicated below:<br /><br />
Total: 10μl<br /><br />
+ 0.5μl of Afl II restriction enzyme, (company :Takara)<br /><br />
+ 1μl of 10XM buffer<br /><br />
+ 1μl of plasmid DNA<br /><br />
+ 1.0μl of 0.01% BSA<br /><br />
+ 6.5μl of ddH2O <br /><br />
3. Electrophorese the total system and observe the lane separation.</p><br />
<br/><br />
<br />
<a name="Amplify"></a> <br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Polymerase Chain Reaction(GFP &amp; RFP) and Electrophoresis</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>GFP and RFP DNA fragments are the insert which need to be ligate to the plasmid mutant-pSB1A3. Do a PCR amplification can get enough quantities for the following reactions.<br /><br />
<strong>Method </strong><br /><br />
1.Prepare the sample reaction as indicated below:<br /><br />
Total: 100μl ( PCR <a name="OLE_LINK37" id="OLE_LINK37"></a><a name="OLE_LINK36" id="OLE_LINK36">amplification</a> of GFP fragments) <br /><br />
+ 1.0μl of Taq DNA polymerase,#EP0402<br /><br />
+ 10μl of 10XTaq buffer <br /><br />
+ 10μl of MgCl2(25mM)<br /><br />
+ 10μl of dNTP(2mM)<br /><br />
+ 2μl of G-SXA-R* <br /><br />
+ 2μl of G-SXA-F*<br /><br />
+ 4μl of DNA template<br /><br />
+ 61μl of ddH2O <br /><br />
Total: 100μl(PCR amplification of RFP fragments) <br /><br />
+ 1.0μl of Taq DNA polymerase, EP0402<br /><br />
+ 10μl of 10XTaq buffer <br /><br />
+ 10μl of MgCl2(25mM)<br /><br />
+ 10μl of dNTP(2mM)<br /><br />
+ 2μl of R-NPS-R* <br /><br />
+ 2μl of R-NPS-F*<br /><br />
+ 4μl of DNA template<br /><br />
+ 61μl of ddH2O <br /><br />
<br />
Note: The sequences of primers R-NPS-F , R-NPS-R , G-SXA-F ,G-SXA-R <br /><br />
R-NPS-F 5'-TATAGCGGCCGCCTTAAGTAAGTAAGAGTATACG-3' <br /><br />
R-NPS-R 5'-CGGAGACTAGTCTGCAGATCACATAAGTAAAGTGATAATC-3' <br /><br />
G-SXA-F 5'-CTAGACTAGTTCTAGAGGCGGACTCACTATAGA-3' <br /><br />
G-SXA-R 5'-TCTCCGACTTAAGGGATCCTATAAACGCAG-3'<br /><br />
<br />
<p> 2. Set parameters for PCR to amplify desired products. </p><br />
<br />
<table><br />
<tr><br />
<td>Temperature</td> <td>Time</td> <td>Cycle</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>5min</td> <td>1</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>55˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>72˚C</td> <td>1min 20sec</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>4˚C</td> <td>7hrs</td> <td>1</td><br />
</tr><br />
</table><br />
</p><br />
<p>3. Use DNA Gel Extraction Kit to purify the GFP and RFP DNA fragments after the Electrophoresis.</p><br />
<br />
<p><font face="Arial, Helvetica"><img src="https://static.igem.org/mediawiki/2012/7/72/111.png" alt="" class="img_fl img_border" align="left"/> </font></p><br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<p>(Figure 2 : This figure shows that The PCR reaction system can amplify large quantities of GFP and RFP DNA fragments. The digestion based on the GFP and RFP DNA fragments can be done to prepare for the ligation. Lane 1 represents the template E.coli 817 can amplify the GFP and RFP DNA fragments , lane 2 represents the template E.coli 817(355.5) can also amplify the GFP and RFP DNA fragments.)<br/><br/><br />
<font face="Arial, Helvetica"><br/><br />
<a name="Restriction_Enzyme"></a><br />
</font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Double Restriction Enzyme Digestion and Electrophoresis.</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Use specific restriction enzymes to digest plasmid mutant-pSB1A3,GFP and RFP to get sticky ends and purify the DNA fragment after the Electrophoresis.<br /></p><br />
<p> <strong>Method:</strong><br/><br />
</font><br />
<ul><br />
<li> Digestion of plasmid mutant-pSB1A3 </li><br />
</ul><br />
<p>Prepare the sample reaction as indicated below:<br /><br />
Total: 50μl <br /><br />
+ 3.0μl of Not I restriction enzyme, #ER0591<br /><br />
+ 3.0μl of Afl II restriction enzyme, #ER0831<br /><br />
+ 5.0μl of 10X buffer O <br /><br />
+ 1.0μl of mutant-pSB1A3 plasmid <br /><br />
+ 37.0μl of ddH2O<br /><br />
2. Digestion of PCR product GFP<br /><br />
Prepare the sample reaction as indicated below:<br /><br />
Total: 50μl <br /><br />
+ 3.0μl of Not I restriction enzyme, #ER0591<br /><br />
+ 3.0μl of Spe I restriction enzyme, #ER1251 <br /><br />
+ 5μl of 10X buffer Tango<br /><br />
+ 10μl of PCR products GFP<br /><br />
+ 29μl of ddH2O<br /><br />
3. Digestion of PCR product RFP<br /><br />
Prepare the sample reaction as indicated below:<br /><br />
Total: 50μl <br /><br />
+ 3.0μl of Afl II restriction enzyme, #ER0831<br /><br />
+ 3.0μl of Spe I restriction enzyme, #ER1251 <br /><br />
+ 5μl of 10X buffer Tango<br /><br />
+ 10μl of PCR products RFP<br /><br />
+ 29μl of ddH2O<br /><br />
4. Put the tubes in 37℃ environment for 4-8 hours <br /><br />
5. Use DNA Gel Extraction Kit to purify the mutant-pSB1A3 fragment, GFP and RFP after digestion and named them by mutant-pSB1A3 (NA) ,GFP(NS) and RFP(AS) after the Electrophoresis.</p><br />
</p><br />
<br />
<font face="Arial, Helvetica"><br><br />
<a name="Ligayion"></a> </font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Ligation</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Ligation is the process that target DNA gene is inserted into a plasmid. Both the vector and insert are prepared to have the sticky ends. These two kinds of DNA pieces are placed in a reaction tube and the proper DNA ligase, buffer, and cofactors are added for the reaction to take place. When done properly, the ligation will result in a successful combination of the insert and plasmid into one plasmid.Based on the digestion of mutant-pSB1A3,GFP and RFP DNA fragments,also ligation can be done. We ligate mutant-pSB1A3 vector and sticky GFP and RFP DNA fragments to construct an new plasmid mutant-pSB1A3-GR. <br /><br />
1. Prepare the control reaction as indicated below:<br /><br />
Total: 10μl <br /><br />
+ 1.0μl of plasmid mutant-pSB1A3 (NA) <br /><br />
+ 3.0μl of GFP(NS) <br /><br />
+ 3.0μl of RFP(AS) <br /><br />
+ 2.0μl of T4 DNA Ligase,#EL0011 <br /><br />
+ 1.0μl of 10XT4 Ligase buffer<br /><br />
Note: GFP(NS) means the product of GFP DNA fragments digested by restriction enzyme Not I and Spe I. <br /><br />
2. Prepare the sample reaction as indicated below:<br /><br />
Total: 10μl <br /><br />
+ 1.0μl of plasmid mutant-pSB1A3 (NA) <br /><br />
+ 2.0μl of T4 DNA Ligase, #EL0011 <br /><br />
+ 1.0μl of 10XT4 Ligase buffer<br /><br />
+ 6.0μl of ddH2O<br /><br />
3. Put the tubes in 22℃ water bath, react for 8-12 hours. <br><br />
<a name="Bacterial_Transformation"></a> </font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Bacterial Transformation</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Transform the ligation products into the DH-5α competent cells, put the plate on 37℃ gas bath for 12-16 hours. </p><br />
<br><br />
<a name="Bacterial_Colony"></a> </font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Bacterial Colony PCR</font></b> </font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Colony PCR is used to identify and select cell colonies that have the correct plasmid insert. This procedure is a way to do several PCR operations on cell colonies in parallel, to evaluate the results and select the corresponding good cell colonies. After an overnight growth of E.coli, we can pick up some colonies from the plate and do a colony PCR verification. Besides, the colonies we choose and should also be stored, we can incubate these colonies in one plate after every colony has been marked.<br /><br />
<strong>Method</strong><br /><br />
1. Prepare the sample reaction as indicated below:<br /><br />
Total: 20μl <br /><br />
+ 0.25 μl of Ex Taq polymerase,#EP0402 <br /><br />
+ 2.0 μl of 10× Taq reaction buffer<br /><br />
+ 1.0 μl of R-NPS-F* <br /><br />
+ 1.0 μl of G-SXA-R*<br /><br />
+ 5.0 μl of bacterial colony<br /><br />
+ 2.0 μl of dNTP( 25mM ) <br /><br />
+ 8.75 μl of ddH2O <br /><br />
Note: The sequences of primers R-NPS-F , R-NPS-R , G-SXA-F ,G-SXA-R <br /><br />
R-NPS-F 5'-TATAGCGGCCGCCTTAAGTAAGTAAGAGTATACG-3' <br /><br />
R-NPS-R 5'-CGGAGACTAGTCTGCAGATCACATAAGTAAAGTGATAATC-3' <br /><br />
G-SXA-F 5'-CTAGACTAGTTCTAGAGGCGGACTCACTATAGA-3' <br /><br />
G-SXA-R 5'-TCTCCGACTTAAGGGATCCTATAAACGCAG-3'<br /><br />
<br />
<p> 2. Set parameters for PCR to amplify desired products. </p> <br />
<table><br />
<tr><br />
<td>Temperature</td> <td>Time</td> <td>Cycle</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>13.5min</td> <td>1</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>55˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>72˚C</td> <td>1min 20sec</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>4˚C</td> <td>7hrs</td> <td>1</td><br />
</tr><br />
</table><br />
<br />
</font><br />
<p> 3. Electrophorese the total system and observe the lane separation. </p><br />
<br />
<p> <img src="https://static.igem.org/mediawiki/2012/0/07/1111.png" alt="" class="img_fl img_border" align="left"/> </p><br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<p>(Figure 3: The lane on the figure are 2k DNA fragments, it shows the GFP and RFP DNA fragments are ligated to the vectors which were isolated from the bacterial colonies.) </p><br />
<br />
<p><font face="Arial, Helvetica"><br><br />
<a name="Culture_the"></a><br />
</font></p><br />
<font face="Arial, Helvetica"><br />
</font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Cultivate the Bacteria</b></font></h3><br />
<font face="Arial, Helvetica"><p>According to the results of the PCR detection, we choose positive colonies and transfer them to 5ml LB liquid media ( 5μl of ampicillin has added) stored in 12.5ml centrifuge tubes. Put the centrifuge tubes in 37℃ gas bath overnight. </p><br />
<br />
<p><font face="Arial, Helvetica"><a name="Plasmid_DNA"></a> </font></p><br />
<font face="Arial, Helvetica"> </font> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Plasmid DNA Isolation</b></font></h3><br />
<p><font face="Arial, Helvetica">Use E.Z.N.A.TM Plasmid Mini I to isolate the constructed plasmid mutant-pSB1A3-GR. </font></p><br />
<br />
<p><font face="Arial, Helvetica"><a name="Restriction_Enzyme" ></a> </font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Restriction Enzyme Digestion and Electrophoresis</b></font></h3><br />
<p>From the last step, we got the certain quantities of isolated plasmids. In this step, we do two restriction enzyme digestion reactions, one to prove that the plasmid is construct correctly ( mutant-pSB1A3-GR ), one to get sticky ends preparing for the ligation.<br /><br />
<strong>Method</strong></p><br />
<p>1.Restriction Enzyme Digestion to prove that plasmid is constructed correctly</p><br />
<br />
<p align="left">1.1. Prepare the sample reaction as indicated below:<br /><br />
Total: 10μl<br /><br />
+ 1.0μl of Not I restriction enzyme,#ER0591<br /><br />
+ 1.0μl of Spe I restriction enzyme,#ER1251 <br /><br />
+ 2.0μl of Buffer Tango( 10X )<br /><br />
+ 1.5μl of plasmid mutant-pSB1A3-GR<br /><br />
+ 14.5μl of ddH2O <br /><br />
1.2. Electrophorese the total system and observe the lane separation. </p><br />
<br />
<p>2. Restriction Enzyme Digestion to get sticky ends preparing for the ligation.</p><br />
<br />
<p align="left">2.1. Prepare the sample reaction as indicated below:<br /><br />
Total: 50μl<br /><br />
+ 5.0μl of Pst I restriction enzyme, #ER0611<br /><br />
+ 5.0μl of Xba I restriction enzyme, #ER0681 <br /><br />
+ 3.0μl of Buffer Tango( 10X )<br /><br />
+ 5.0μl of plasmid mutant-pSB1A3-GR<br /><br />
+ 32.0μl of ddH2O <br /><br />
2.2. Electrophorese the total system and observe the lane separation.<br /><br />
2.3. Cut the gel of specific position and collect it in tubes that have measured weight. <br /><br />
2.4. Use DNA Gel Extraction Ki to purify the plasmid DNA mutant-pSB1A3-GR(PX) .<br /><br />
Note: Mutant-pSB1A3-GR(PX) means plasmid Mutant-pSB1A3-GR digested by Pst I and Xba I.</p><br />
<br />
<br />
<p align="left"><strong><img src="https://static.igem.org/mediawiki/2012/d/d8/11111.png" alt="" class="img_fl img_border" align="left" /></strong></p><br />
<p>(Figure 4 : The double digestion of mutant-pSB1A3 forms a linear DNA fragments and it runs slower than circle DNA fragments. This suggest that the double digestion of mutant-pSB1A3 works in a high efficiency, and desired sticky ends are formed.1,3,5 are plasmids digested by restriction enzyme Pst I and Xba I from different colonies, 2,5,6 are pure plasmid mutant-pSB1A3-GR, 7 is the plasmid mutant-pSB1A3. )</p><br />
<p align="left">&nbsp;</p><br />
<p><font face="Arial, Helvetica"><a name="Ligation1" ></a> </font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Ligation</b></font></h3><br />
<p>Ligation is the process that target DNA gene is inserted into a plasmid. Both the vector and insert are prepared to have the sticky ends. These two kinds of DNA pieces are placed in a reaction tube and the proper DNA ligase, buffer, and cofactors are added for the reaction to take place. When done properly, the ligation will result in a successful combination of the insert and plasmid into one plasmid.Based on the digestion of mutant-pSB1A3-GR, terminator DNA fragments,ligation can be done. We ligate mutant-pSB1A3-GR vector and sticky terminator DNA fragments to construct an new plasmid mutant-pSB1A3-GR-t.By detecting the quantities of GFP and RFP, terminator efficiency can be calculated. <br /><br />
1. Prepare the control reaction as indicated below:<br /><br />
Total: 10μl <br /><br />
+ 1.0μl of plasmid mutant-pSB1A3 (NA) <br /><br />
+ 6.0μl of terminator<br /><br />
+ 2.0μl of T4 DNA Ligase , #EL0011 <br /><br />
+ 1.0μl of 10XT4 Ligase buffer<br /><br />
2. Put the tubes in 22℃ water bath, react for 8-12 hours. </p><br />
<p><font face="Arial, Helvetica"><a name="Bacteria" id="Culture_the"></a> </font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Bacteria transformation</b></font></h3><br />
<p>Transform the ligation products into the DH-5α competent cells, put the plate on 37℃ gas bath for 12-16 hours. </p><br />
<p><font face="Arial, Helvetica"><a name="Cultivate"></a> </font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Cultivate the Bacteria</b></font></h3><br />
<p>According to the results of the PCR detection, we choose positive colonies and transfer them to 5ml LB liquid media ( 5μl of ampicillin has added) stored in 12.5ml centrifuge tubes. Put the centrifuge tubes in 37℃ gas bath overnight. </p><br />
<br />
<p><font face="Arial, Helvetica"><a name="Flow" ></a> </font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Flow Cytometer Analysis</b></font></h3><br />
<p>Flow cytometry is a laser based, biophysical technology employed in cell counting, sorting, biomarker detection and protein engineering, by suspending cells in a stream of fluid and passing them by an electronic detection apparatus. It allows simultaneous multiparametric analysis of the physical and/or chemical characteristics of up to thousands of particles per second. </p><br />
<p>Each fluorophore has a characteristic peak excitation and emission wavelength, and the emission spectra often overlap. Consequently, the combination of labels which can be used depends on the wavelength of the lamp(s) or laser(s) used to excite the fluorochromes and on the detectors available. It is practical that the expression of RFP and GFP can be measured at the same time.</p><br />
<p><strong>Method:</strong><br/><br />
1. Materials:<br/><br />
&nbsp;&nbsp;1.1. NaCl solution( 0.9% ,M/V)<br/><br />
&nbsp;&nbsp;1.2. 75% ethanol<br/><br />
2. Procedures:<br/><br />
&nbsp;&nbsp;2.1. Bacterium are harvested by centrifuge at 10000g for 30s, then discard the supernatant. Repeat this step again until we got adequate bacterium.<br/><br />
&nbsp;&nbsp;2.2. Suspend the bacterium With NaCl solution(0.9%) and vibrate the centrifuge tubes until the bacterium are distributed homogeneous.<br/><br />
&nbsp;&nbsp;2.3. Load the bacteria containing pSB1A3 to Flow Cytometer (Beckman), set the parameters. Measure the GFP-FL1 and RFP-FL2 by Cytometer. </p><br />
<br />
<p><font face="Arial, Helvetica"><a name="Fluorescence"></a> </font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Fluorescence Microscope</b></font></h3><br />
<p>A fluorescence microscope is an optical microscope that uses fluorescence and phosphorescence reflection and absorption to study properties of organic or inorganic substances and generate an image.Fluorescence microscope detection are used to confirm the expression of GFP and RFP which are illuminated with light of a wavelength which excites fluorescence in the sample.<br />
</p><br />
<p><strong>Method</strong><br /><br />
Add 10μl of bacteria solution to micro slide and cover with coverslip.<br />
Placed the micro slide on the Fluorescence Microscope. Set parameters to detect GFP and RFP. <br/><br />
Generate an image of fluorescence protein and save it.</p><br />
<br />
<br />
<br> <br />
<br />
</article><br />
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</html></div>M.B.ZHOUhttp://2012.igem.org/Team:SUSTC-Shenzhen-B/protocol1Team:SUSTC-Shenzhen-B/protocol12012-10-26T20:54:31Z<p>M.B.ZHOU: </p>
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<meta name="description" content="" /><br />
<meta name="author" content="tengattack" /><br />
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Sans',sans-serif;-webkit-border-top-left-radius:3px;-webkit-border-top-right-radius:3px;-moz-border-radius-topleft:3px;-moz-border-radius-topright:3px;border-top-left-radius:3px;border-top-right-radius:3px}.tab-content{padding:20px 0}ul.tabs li:hover{}ul.tabs li.active{}html ul.tabs li.active a{color:#b03121;background:#e1dfc9;-webkit-border-top-left-radius:3px;-webkit-border-top-right-radius:3px;-moz-border-radius-topleft:3px;-moz-border-radius-topright:3px;border-top-left-radius:3px;border-top-right-radius:3px}#tab-body{padding:0 20px;background:#e1dfc9;border:0;-webkit-border-radius:3px;-webkit-border-top-left-radius:0;-moz-border-radius:3px;-moz-border-radius-topleft:0;border-radius:3px;border-top-left-radius:0}#toggle{margin:0 0 20px 0}h2.trigger{padding:0 0;margin:0;font-size:13px;background:transparent;font-family:arial;color:#3f4432}h2.trigger span{text-decoration:none;display:block;background:url(/wiki/images/9/93/Sustc-b1-toggle.png) no-repeat 0 5px;padding-left:35px;cursor:pointer;line-height:35px}h2.active span{background:url(/wiki/images/c/cd/Sustc-b1-toggle-down.png) no-repeat 0 5px}h2.trigger a:hover{color:#efeed9}h2.active{background:transparent;color:#af3728}.toggle_container{margin:0 0 1px 0;padding:5px 15px;overflow:hidden;clear:both;background:transparent}.toggle_container .block{padding:0 0 0 20px}.toggle_container .block p{padding-bottom:10px;margin:0}#sidebar{width:300px;float:left;padding:0 0 0 40px}#sidebar.positionleft{float:left;padding:0 40px 0 0}#sidebar.positionright{float:right}#sidebar .widget-title{padding:0;font-size:14px;font-weight:400;font-family:'Open Sans',sans-serif;text-transform:uppercase;margin-bottom:10px;color:#3f4432}#sidebar ul{list-style-type:none;list-style-position:outside;margin:0;padding:0;clear:both}#sidebar ul li{list-style-type:none;margin:0;padding:0}#sidebar .widget-container{margin-bottom:28px;padding-top:28px;background:url(/wiki/images/7/79/Sustc-b1-line.gif) no-repeat}#sidebar .widget-container:first-child{background:0;padding:0}#sidebar li li{list-style-type:none;margin:0 0 3px 0;padding:0 0 3px 0}#sidebar li li a{color:#777}#sidebar li li a:hover{text-decoration:none;color:#af3728}#sidebar ul.sub-menu,#sidebar ul.children,#sidebar ul ul ul{margin:5px 0 0 10px}#sidebar ul.sub-menu li,#sidebar ul.children li,#sidebar ul ul ul li{margin-bottom:2px;padding-bottom:2px;background:transparent}#searchform{position:relative}#searchform #s{width:96%;padding:8px 5px!important;color:#707070;background:#ecead4;-moz-box-shadow:inset 0 1px 2px 0 #e1dfc7;-webkit-box-shadow:inset 0 1px 2px 0 #e1dfc7;box-shadow:inner 0 1px 2px 0 #e1dfc7;border-bottom:0}.rp-widget li{clear:left;margin-bottom:0;padding-bottom:10px}.rp-widget img{padding:4px}.rp-widget li h3{margin-bottom:0}.rp-widget li h3 a{font-size:13px;font-weight:600}.rp-widget li .smalldate{display:block;font-size:11px;font-style:italic;overflow:hidden}#footercontainer{background:#3f4432;-webkit-border-bottom-left-radius:7px;-webkit-border-bottom-right-radius:7px;-moz-border-radius-bottomleft:7px;-moz-border-radius-bottomright:7px;border-bottom-left-radius:7px;border-bottom-right-radius:7px}#footer{padding:25px 0 25px 0;color:#bbb9a1;font-weight:400;font-family:'Open Sans',sans-serif;font-size:13px}#footer a,#footer a:visited{color:#bbb9a1}<br />
<br />
/* prettyPhoto.css */<br />
div.pp_default .pp_top,div.pp_default .pp_top .pp_middle,div.pp_default .pp_top .pp_left,div.pp_default .pp_top .pp_right,div.pp_default .pp_bottom,div.pp_default .pp_bottom .pp_left,div.pp_default .pp_bottom .pp_middle,div.pp_default .pp_bottom .pp_right{height:13px}div.pp_default .pp_top .pp_left{background:url(../images/default/sprite.png) -78px -93px no-repeat}div.pp_default .pp_top .pp_middle{background:url(../images/default/sprite_x.png) top left repeat-x}div.pp_default .pp_top .pp_right{background:url(../images/default/sprite.png) -112px -93px no-repeat}div.pp_default .pp_content .ppt{color:#f8f8f8}div.pp_default .pp_content_container .pp_left{background:url(../images/default/sprite_y.png) -7px 0 repeat-y;padding-left:13px}div.pp_default .pp_content_container .pp_right{background:url(../images/default/sprite_y.png) top right repeat-y;padding-right:13px}div.pp_default .pp_next:hover{background:url(../images/default/sprite_next.png) center right no-repeat;cursor:pointer}div.pp_default .pp_previous:hover{background:url(../images/default/sprite_prev.png) center left no-repeat;cursor:pointer}div.pp_default .pp_expand{background:url(../images/default/sprite.png) 0 -29px no-repeat;cursor:pointer;height:28px;width:28px}div.pp_default .pp_expand:hover{background:url(../images/default/sprite.png) 0 -56px no-repeat;cursor:pointer}div.pp_default .pp_contract{background:url(../images/default/sprite.png) 0 -84px no-repeat;cursor:pointer;height:28px;width:28px}div.pp_default .pp_contract:hover{background:url(../images/default/sprite.png) 0 -113px no-repeat;cursor:pointer}div.pp_default .pp_close{background:url(../images/default/sprite.png) 2px 1px no-repeat;cursor:pointer;height:30px;width:30px}div.pp_default .pp_gallery ul li a{background:url(../images/default/default_thumb.png) center center #f8f8f8;border:1px solid #aaa}div.pp_default .pp_social{margin-top:7px}div.pp_default .pp_gallery a.pp_arrow_previous,div.pp_default .pp_gallery a.pp_arrow_next{left:auto;position:static}div.pp_default .pp_nav .pp_play,div.pp_default .pp_nav .pp_pause{background:url(../images/default/sprite.png) -51px 1px no-repeat;height:30px;width:30px}div.pp_default .pp_nav .pp_pause{background-position:-51px -29px}div.pp_default a.pp_arrow_previous,div.pp_default a.pp_arrow_next{background:url(../images/default/sprite.png) -31px -3px no-repeat;height:20px;margin:4px 0 0;width:20px}div.pp_default a.pp_arrow_next{background-position:-82px -3px;left:52px}div.pp_default .pp_content_container .pp_details{margin-top:5px}div.pp_default .pp_nav{clear:none;height:30px;position:relative;width:110px}div.pp_default .pp_nav .currentTextHolder{color:#999;font-family:Georgia;font-size:11px;font-style:italic;left:75px;line-height:25px;margin:0;padding:0 0 0 10px;position:absolute;top:2px}div.pp_default .pp_close:hover,div.pp_default .pp_nav .pp_play:hover,div.pp_default .pp_nav .pp_pause:hover,div.pp_default .pp_arrow_next:hover,div.pp_default .pp_arrow_previous:hover{opacity:.7}div.pp_default .pp_description{font-size:11px;font-weight:700;line-height:14px;margin:5px 50px 5px 0}div.pp_default .pp_bottom .pp_left{background:url(../images/default/sprite.png) -78px -127px no-repeat}div.pp_default .pp_bottom .pp_middle{background:url(../images/default/sprite_x.png) bottom left repeat-x}div.pp_default .pp_bottom .pp_right{background:url(../images/default/sprite.png) -112px -127px no-repeat}div.pp_default .pp_loaderIcon{background:url(../images/default/loader.gif) center center no-repeat}div.light_rounded .pp_top .pp_left{background:url(../images/light_rounded/sprite.png) -88px -53px no-repeat}div.light_rounded .pp_top .pp_right{background:url(../images/light_rounded/sprite.png) -110px -53px no-repeat}div.light_rounded .pp_next:hover{background:url(../images/light_rounded/btnNext.png) center right no-repeat;cursor:pointer}div.light_rounded .pp_previous:hover{background:url(../images/light_rounded/btnPrevious.png) center left no-repeat;cursor:pointer}div.light_rounded .pp_expand{background:url(../images/light_rounded/sprite.png) -31px -26px no-repeat;cursor:pointer}div.light_rounded .pp_expand:hover{background:url(../images/light_rounded/sprite.png) -31px -47px no-repeat;cursor:pointer}div.light_rounded .pp_contract{background:url(../images/light_rounded/sprite.png) 0 -26px no-repeat;cursor:pointer}div.light_rounded .pp_contract:hover{background:url(../images/light_rounded/sprite.png) 0 -47px no-repeat;cursor:pointer}div.light_rounded .pp_close{background:url(../images/light_rounded/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:75px}div.light_rounded .pp_nav .pp_play{background:url(../images/light_rounded/sprite.png) -1px -100px no-repeat;height:15px;width:14px}div.light_rounded .pp_nav .pp_pause{background:url(../images/light_rounded/sprite.png) -24px -100px no-repeat;height:15px;width:14px}div.light_rounded .pp_arrow_previous{background:url(../images/light_rounded/sprite.png) 0 -71px no-repeat}div.light_rounded .pp_arrow_next{background:url(../images/light_rounded/sprite.png) -22px -71px no-repeat}div.light_rounded .pp_bottom .pp_left{background:url(../images/light_rounded/sprite.png) -88px -80px no-repeat}div.light_rounded .pp_bottom .pp_right{background:url(../images/light_rounded/sprite.png) -110px -80px no-repeat}div.dark_rounded .pp_top .pp_left{background:url(../images/dark_rounded/sprite.png) -88px -53px no-repeat}div.dark_rounded .pp_top .pp_right{background:url(../images/dark_rounded/sprite.png) -110px -53px no-repeat}div.dark_rounded .pp_content_container .pp_left{background:url(../images/dark_rounded/contentPattern.png) top left repeat-y}div.dark_rounded .pp_content_container .pp_right{background:url(../images/dark_rounded/contentPattern.png) top right repeat-y}div.dark_rounded .pp_next:hover{background:url(../images/dark_rounded/btnNext.png) center right no-repeat;cursor:pointer}div.dark_rounded .pp_previous:hover{background:url(../images/dark_rounded/btnPrevious.png) center left no-repeat;cursor:pointer}div.dark_rounded .pp_expand{background:url(../images/dark_rounded/sprite.png) -31px -26px no-repeat;cursor:pointer}div.dark_rounded .pp_expand:hover{background:url(../images/dark_rounded/sprite.png) -31px -47px no-repeat;cursor:pointer}div.dark_rounded .pp_contract{background:url(../images/dark_rounded/sprite.png) 0 -26px no-repeat;cursor:pointer}div.dark_rounded .pp_contract:hover{background:url(../images/dark_rounded/sprite.png) 0 -47px no-repeat;cursor:pointer}div.dark_rounded .pp_close{background:url(../images/dark_rounded/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:75px}div.dark_rounded .pp_description{color:#fff;margin-right:85px}div.dark_rounded .pp_nav .pp_play{background:url(../images/dark_rounded/sprite.png) -1px -100px no-repeat;height:15px;width:14px}div.dark_rounded .pp_nav .pp_pause{background:url(../images/dark_rounded/sprite.png) -24px -100px no-repeat;height:15px;width:14px}div.dark_rounded .pp_arrow_previous{background:url(../images/dark_rounded/sprite.png) 0 -71px no-repeat}div.dark_rounded .pp_arrow_next{background:url(../images/dark_rounded/sprite.png) -22px -71px no-repeat}div.dark_rounded .pp_bottom .pp_left{background:url(../images/dark_rounded/sprite.png) -88px -80px no-repeat}div.dark_rounded .pp_bottom .pp_right{background:url(../images/dark_rounded/sprite.png) -110px -80px no-repeat}div.dark_rounded .pp_loaderIcon{background:url(../images/dark_rounded/loader.gif) center center no-repeat}div.dark_square .pp_left,div.dark_square .pp_middle,div.dark_square .pp_right,div.dark_square .pp_content{background:#000}div.dark_square .pp_description{color:#fff;margin:0 85px 0 0}div.dark_square .pp_loaderIcon{background:url(../images/dark_square/loader.gif) center center no-repeat}div.dark_square .pp_expand{background:url(../images/dark_square/sprite.png) -31px -26px no-repeat;cursor:pointer}div.dark_square .pp_expand:hover{background:url(../images/dark_square/sprite.png) -31px -47px no-repeat;cursor:pointer}div.dark_square .pp_contract{background:url(../images/dark_square/sprite.png) 0 -26px no-repeat;cursor:pointer}div.dark_square .pp_contract:hover{background:url(../images/dark_square/sprite.png) 0 -47px no-repeat;cursor:pointer}div.dark_square .pp_close{background:url(../images/dark_square/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:75px}div.dark_square .pp_nav{clear:none}div.dark_square .pp_nav .pp_play{background:url(../images/dark_square/sprite.png) -1px -100px no-repeat;height:15px;width:14px}div.dark_square .pp_nav .pp_pause{background:url(../images/dark_square/sprite.png) -24px -100px no-repeat;height:15px;width:14px}div.dark_square .pp_arrow_previous{background:url(../images/dark_square/sprite.png) 0 -71px no-repeat}div.dark_square .pp_arrow_next{background:url(../images/dark_square/sprite.png) -22px -71px no-repeat}div.dark_square .pp_next:hover{background:url(../images/dark_square/btnNext.png) center right no-repeat;cursor:pointer}div.dark_square .pp_previous:hover{background:url(../images/dark_square/btnPrevious.png) center left no-repeat;cursor:pointer}div.light_square .pp_expand{background:url(../images/light_square/sprite.png) -31px -26px no-repeat;cursor:pointer}div.light_square .pp_expand:hover{background:url(../images/light_square/sprite.png) -31px -47px no-repeat;cursor:pointer}div.light_square .pp_contract{background:url(../images/light_square/sprite.png) 0 -26px no-repeat;cursor:pointer}div.light_square .pp_contract:hover{background:url(../images/light_square/sprite.png) 0 -47px no-repeat;cursor:pointer}div.light_square .pp_close{background:url(../images/light_square/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:75px}div.light_square .pp_nav .pp_play{background:url(../images/light_square/sprite.png) -1px -100px no-repeat;height:15px;width:14px}div.light_square .pp_nav .pp_pause{background:url(../images/light_square/sprite.png) -24px -100px no-repeat;height:15px;width:14px}div.light_square .pp_arrow_previous{background:url(../images/light_square/sprite.png) 0 -71px no-repeat}div.light_square .pp_arrow_next{background:url(../images/light_square/sprite.png) -22px -71px no-repeat}div.light_square .pp_next:hover{background:url(../images/light_square/btnNext.png) center right no-repeat;cursor:pointer}div.light_square .pp_previous:hover{background:url(../images/light_square/btnPrevious.png) center left no-repeat;cursor:pointer}div.facebook .pp_top .pp_left{background:url(../images/facebook/sprite.png) -88px -53px no-repeat}div.facebook .pp_top .pp_middle{background:url(../images/facebook/contentPatternTop.png) top left repeat-x}div.facebook .pp_top .pp_right{background:url(../images/facebook/sprite.png) -110px -53px no-repeat}div.facebook .pp_content_container .pp_left{background:url(../images/facebook/contentPatternLeft.png) top left repeat-y}div.facebook .pp_content_container .pp_right{background:url(../images/facebook/contentPatternRight.png) top right repeat-y}div.facebook .pp_expand{background:url(../images/facebook/sprite.png) -31px -26px no-repeat;cursor:pointer}div.facebook .pp_expand:hover{background:url(../images/facebook/sprite.png) -31px -47px no-repeat;cursor:pointer}div.facebook .pp_contract{background:url(../images/facebook/sprite.png) 0 -26px no-repeat;cursor:pointer}div.facebook .pp_contract:hover{background:url(../images/facebook/sprite.png) 0 -47px no-repeat;cursor:pointer}div.facebook .pp_close{background:url(../images/facebook/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:22px}div.facebook .pp_description{margin:0 37px 0 0}div.facebook .pp_loaderIcon{background:url(../images/facebook/loader.gif) center center no-repeat}div.facebook .pp_arrow_previous{background:url(../images/facebook/sprite.png) 0 -71px no-repeat;height:22px;margin-top:0;width:22px}div.facebook .pp_arrow_previous.disabled{background-position:0 -96px;cursor:default}div.facebook .pp_arrow_next{background:url(../images/facebook/sprite.png) -32px -71px no-repeat;height:22px;margin-top:0;width:22px}div.facebook .pp_arrow_next.disabled{background-position:-32px -96px;cursor:default}div.facebook .pp_nav{margin-top:0}div.facebook .pp_nav p{font-size:15px;padding:0 3px 0 4px}div.facebook .pp_nav .pp_play{background:url(../images/facebook/sprite.png) -1px -123px no-repeat;height:22px;width:22px}div.facebook .pp_nav .pp_pause{background:url(../images/facebook/sprite.png) -32px -123px no-repeat;height:22px;width:22px}div.facebook .pp_next:hover{background:url(../images/facebook/btnNext.png) center right no-repeat;cursor:pointer}div.facebook .pp_previous:hover{background:url(../images/facebook/btnPrevious.png) center left no-repeat;cursor:pointer}div.facebook .pp_bottom .pp_left{background:url(../images/facebook/sprite.png) -88px -80px no-repeat}div.facebook .pp_bottom .pp_middle{background:url(../images/facebook/contentPatternBottom.png) top left repeat-x}div.facebook .pp_bottom .pp_right{background:url(../images/facebook/sprite.png) -110px -80px no-repeat}div.pp_pic_holder a:focus{outline:0}div.pp_overlay{background:#000;display:none;left:0;position:absolute;top:0;width:100%;z-index:9500}div.pp_pic_holder{display:none;position:absolute;width:100px;z-index:10000}.pp_content{height:40px;min-width:40px}* html .pp_content{width:40px}.pp_content_container{position:relative;text-align:left;width:100%}.pp_content_container .pp_left{padding-left:20px}.pp_content_container .pp_right{padding-right:20px}.pp_content_container .pp_details{float:left;margin:10px 0 2px}.pp_description{display:none;margin:0}.pp_social{float:left;margin:0}.pp_social .facebook{float:left;margin-left:5px;overflow:hidden;width:55px}.pp_social .twitter{float:left}.pp_nav{clear:right;float:left;margin:3px 10px 0 0}.pp_nav p{float:left;margin:2px 4px;white-space:nowrap}.pp_nav .pp_play,.pp_nav .pp_pause{float:left;margin-right:4px;text-indent:-10000px}a.pp_arrow_previous,a.pp_arrow_next{display:block;float:left;height:15px;margin-top:3px;overflow:hidden;text-indent:-10000px;width:14px}.pp_hoverContainer{position:absolute;top:0;width:100%;z-index:2000}.pp_gallery{display:none;left:50%;margin-top:-50px;position:absolute;z-index:10000}.pp_gallery div{float:left;overflow:hidden;position:relative}.pp_gallery ul{float:left;height:35px;margin:0 0 0 5px;padding:0;position:relative;white-space:nowrap}.pp_gallery ul a{border:1px rgba(0,0,0,.5) solid;display:block;float:left;height:33px;overflow:hidden}.pp_gallery ul a img{border:0}.pp_gallery li{display:block;float:left;margin:0 5px 0 0;padding:0}.pp_gallery li.default a{background:url(../images/facebook/default_thumbnail.gif) 0 0 no-repeat;display:block;height:33px;width:50px}.pp_gallery .pp_arrow_previous,.pp_gallery 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.pp_content{background-color:#fff}div.pp_default #pp_full_res .pp_inline,div.light_rounded .pp_content .ppt,div.light_rounded #pp_full_res .pp_inline,div.light_square .pp_content .ppt,div.light_square #pp_full_res .pp_inline,div.facebook .pp_content .ppt,div.facebook #pp_full_res .pp_inline{color:#000}div.pp_default .pp_gallery ul li a:hover,div.pp_default .pp_gallery ul li.selected a,.pp_gallery ul a:hover,.pp_gallery li.selected a{border-color:#fff}div.pp_default .pp_details,div.light_rounded .pp_details,div.dark_rounded .pp_details,div.dark_square .pp_details,div.light_square .pp_details,div.facebook .pp_details{position:relative}div.light_rounded .pp_top .pp_middle,div.light_rounded .pp_content_container .pp_left,div.light_rounded .pp_content_container .pp_right,div.light_rounded .pp_bottom .pp_middle,div.light_square .pp_left,div.light_square .pp_middle,div.light_square .pp_right,div.light_square .pp_content,div.facebook .pp_content{background:#fff}div.light_rounded 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/*<br />
* Superfish v1.4.8 - jQuery menu widget<br />
* Copyright (c) 2008 Joel Birch<br />
*<br />
* Dual licensed under the MIT and GPL licenses:<br />
* http://www.opensource.org/licenses/mit-license.php<br />
* http://www.gnu.org/licenses/gpl.html<br />
*<br />
* CHANGELOG: http://users.tpg.com.au/j_birch/plugins/superfish/changelog.txt<br />
*/<br />
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/*<br />
* Supersubs v0.2b - jQuery plugin<br />
* Copyright (c) 2008 Joel Birch<br />
*<br />
* Dual licensed under the MIT and GPL licenses:<br />
* http://www.opensource.org/licenses/mit-license.php<br />
* http://www.gnu.org/licenses/gpl.html<br />
*<br />
*<br />
* This plugin automatically adjusts submenu widths of suckerfish-style menus to that of<br />
* their longest list item children. If you use this, please expect bugs and report them<br />
* to the jQuery Google Group with the word 'Superfish' in the subject line.<br />
*<br />
*/<br />
<br />
(function($){ // $ will refer to jQuery within this closure<br />
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};<br />
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// custom.js<br />
jQuery(document).ready(function(){<br />
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//Add Class Js to html<br />
jQuery('html').addClass('js'); <br />
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jQuery("ul.sf-menu").supersubs({ <br />
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extraWidth : 3 // extra width can ensure lines don't sometimes turn over due to slight browser differences in how they round-off values<br />
// due to slight rounding differences and font-family <br />
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// not display:none when measuring. Call before initialising <br />
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jQuery(".tab-content:first").show(); //Show first tab content<br />
//On Click Event<br />
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jQuery(".tab-content").hide(); //Hide all tab content<br />
var activeTab = jQuery(this).find("a").attr("href"); //Find the rel attribute value to identify the active tab + content<br />
jQuery(activeTab).fadeIn(200); //Fade in the active content<br />
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//jQuery toggle<br />
jQuery(".toggle_container").hide();<br />
jQuery("h2.trigger").click(function(){<br />
jQuery(this).toggleClass("active").next().slideToggle("slow");<br />
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<script type="text/javascript"><br />
/* load png for javascript need canvas (HTML5)*/<br />
function png2js(pngurl, callback){<br />
var canvas = document.createElement("canvas"),<br />
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/* jQuery Cycle Plugin (with Transition Definitions) */<br />
png2js("/wiki/images/8/85/Jquery-cycle-all-min-js.png", function() {<br />
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<!-- MAIN CONTENT --><br />
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<div id="maincontent"><br />
<section id="mainthecontent"><br />
<br />
<article><br />
<h1><p>Lab Protocol</p></h1><br />
</font><br />
<font face="Arial, Helvetica"><br />
<p><a href="#Site-Directed_Mutagenesis">1. Site-Directed Mutagenesis</a></p><br />
<p><a href="#Restriction">2. Mutation Verification by Restriction Enzyme Digestion</a></p><br />
<p><a href="#Media">3. Media Preparation</a></p><br />
<p><a href="#Bacterial">4. Bacterial Transformation</a></p><br />
<p><a href="#Colony">5. Colony PCR for Verification</a></p><br />
<p><a href="#Culture">6. Cultivate the Bacteria</a></p><br />
<p><a href="#Plasmid">7. Plasmid DNA Isolation</a></p><br />
<p><a href="#Restriction_2">8. Mutation Verification by Restriction Enzyme Digestion </a></p><br />
<p><a href="#Amplify">9. Polymerase Chain Reaction and Electrophoresis</a></p><br />
<p><a href="#Electrophoresis">10. Double Restriction Enzyme Digestion and Electrophoresis </a></p><br />
<p><a href="#Ligation">11. Ligation</a></p><br />
<p><a href="#Bacterial_Transformation">12. Bacterial Transformation</a></p><br />
<p><a href="#bacterial_colony">13. Bacterial Colony PCR</a></p><br />
<p><a href="#Culture_the">14. Cultivate the Bacteria</a></p><br />
<p><a href="#Plasmid_DNA">15. Plasmid DNA Isolation</a></p><br />
<p><a href="#Restriction_Enzyme">16. Restriction Enzyme Digestion and Electrophoresis</a></p><br />
<p><a href="#Ligation1">17. Ligation</a></p><br />
<p><a href="#Bacteria">18. Bacteria Transformation</a></p><br />
<p><a href="#Cultivate">19. Cultivate the Bacteria</a></p><br />
<p><a href="#Flow">20. Flow Cytometer Analysis</a></p><br />
<p><a href="#Fluorescence">21. Fluorescence Microscope </a></p><br />
<br><br />
<a name="Site-Directed_Mutagenesis" ></a></font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Site-Directed Mutagenesis</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Plasmid pSB1A3 was chosen as a backbone vector for cloning. The Pst I site in pSB1A3 was mutated to Afl II site to facilitate following cloning processes. Proper primers were designed and PCR-based site-directed mutageneis were carried out to generate this mutation as descbibed below. </p><br />
<br />
<p><!--要到很后面才能找到他的结束--><br />
<strong>Method:</strong><br/><br />
<br />
<p>1. Set up PCR assay tubes as described below:<br />
<br/><br />
Total: 25 μl<br /><br />
+ 0.25 μl of Ex Taq polymerase <br /><br />
+ 2.5 μl of 10× Taq reaction buffer<br /><br />
+ 2.0 μl of dNTP(2 mM) <br /><br />
+ 1.0 μl of template (E.coli plasmid 817) <br /><br />
+ 1.0 μl of oligonucleotide primer PtoA-F*<br /><br />
+ 1.0 μl of oligonucleotide primer PtoA-R* <br /><br />
+ 18.25 μl of ddH2O <br /><br />
*The sequences of primer pair PtoA-F and PtoA-R.<br /><br />
PtoA-F 5'-CCACCTGACGTCTAAGAAAC-3'<br /><br />
PtoA-R 5'-ATGATCATCGCCGGCGAATTCAGGC-3' <br /><br />
</p><br />
<p> 2. Set parameters for PCR to amplify desired products. </p><br />
<br />
<table><br />
<tr><br />
<td>Temperature</td> <td>Time</td> <td>Cycle</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>5min</td> <td>1</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>55˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>72˚C</td> <td>1min 20sec</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>4˚C</td> <td>7hrs</td> <td>1</td><br />
</tr><br />
</table><br />
</p><br />
</p><!--那个p的结束,下同--><br />
<br />
<font face="Arial, Helvetica"><br />
<p><br><br />
<a name="Restriction" ></a> </p><br />
</font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Restriction Enzyme Digestion for Verification</font></b></font></h3><br />
<p align="left">To verify whether PstI site in pSB1A3 was successfully mutated to AflII site, we performed restriction enzyme digestion experiments. </p><br />
<p>Bacterial plasmids are double-stranded circular DNA molecules and uncut plasmid DNA can be in any of three forms - nicked circular, linear, closed supercoiled. When run on an agarose gel one frequently will see these forms as different bands with closed supercoiled form migrates the fastest, linear form migrates the slowest, and nicked circular migrates in between. If the PStI site was successfully mutated to AflII site, we expected to see increased linear form of pSB1A3 when digested with AflII. SpeI-cut pSB1A3 was served as a positive control. </p><br /><br />
<p><br />
<strong>Method</strong><br /><br />
1. Set up Pst I digestion in 1.5 ml eppendorf tubes as described below:<br /><br />
Total: 10μl<br /><br />
+ 0.5μl of Pst I restriction enzyme (company :Takara)<br /><br />
+ 1μl of 10XH buffer<br /><br />
+ 1μl of plasmid DNA<br /><br />
+ 7.5μl of ddH2O <br /><br />
2. Set up Afl II digestion in 1.5 ml eppendorf tubes as described below:<br /><br />
Total: 10μl<br /><br />
+ 0.5μl of Afl II restriction enzyme, (company :Takara)<br /><br />
+ 1μl of 10XM buffer<br /><br />
+ 1μl of plasmid DNA<br /><br />
+ 1.0μl of 0.01% BSA<br /><br />
+ 6.5μl of ddH2O<br /><br />
3. Incubate the eppendorf tubes in 37℃ water bath for 1-2 hr. <br/><br />
4. Prepare 1% agarose gel in Conical flask. Weigh 0.6 g agarose and add 60 ml 1x TAE (diluted from 50x TAE). Cover the Conical flask with silver paper to avoid the loss of water vapor. Place the Conical flask in the microwave and microwave for 1 minute with a middle power. Take it out and shake gently till the solution is homogeneous,(BE CAREFUL to watch the solution closely when shake it–it superheats and can boil over and cause severe burns). Continue microwave and swirl until solution is seen clear and homogeneous with no existence of solid. After cool down the agarose gel briefly, add 3 μl of Gelred (10000x ) and mix well. Pour the agarose gel in gel casting apparatus and insert combs.<br/><br />
5. By inserting the pipette tip below the TAE liquid and into the well, add 5 μl of 1kb DNA ladder solution to first (and last if desired) well, skip one well, then begin adding the 5μl of digested DNA solutions mixed with 1 μl loading buffer (6x) to the wells. <br/><br />
6. Place the cover on the electrophoresis unit, plug into the power source, and turn on voltage to 120V, set time to 30 minutes, and press the start button twice,until the bubbles are seen. DNA separation can be observed as time goes on by turning off the power supply then gently removing the basin from the electrophoresis unit (be careful not to let the gel slip out of the basin) and placing on the UV transilluminator to see DNA bands. <br/><br />
7. When the desired level of separation is obtained, the basin can be placed on the transilluminator for picture taking(Of the absence of transilluminator,we use camera to take pictures with the UV light ). <br/><br />
8. Cut the gel of specific position and collect it in tubes that have measured weight. <br/><br />
9. Use DNA Gel Extraction Ki to purify the plasmid DNA mutant-psb1a3.<br />
RPF (SpeI/AflII-digested), GFP (SpeI/AflII-digested) fragments were ligated into this vector, which was followed by insertion of designed terminator sequences between RFP and GFP, respectively.</p><br />
<font face="Arial, Helvetica"><br />
<p> <img src="https://static.igem.org/mediawiki/2012/5/59/11.png" alt="" class="img_fl img_border" align="left"/> </p><br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<p>(Figure 1 : This figure shows that the site-directed mutagenesis succeed ,we successfully change a restriction enzyme cutting site named Pst I to Afl II. Lane 1 represents the plasmid mutant-psb1a3, lane 2 shows that the mutant-psb1a3 cannot be digested by restriction enzyme Spe I ,lane 3 shows that mutant-psb1a3 can be digested by restriction enzyme Afl II.) </p><br />
</p><br />
<br />
<a name="Media"></a></font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Media Preparation</font></b></font></h3><br />
<p align="left">For all experiments involving the bacterial biomass and experimentation, proper media is chosen to grow the cells. Commonly,we use Lysogeny broth media for <em>E. coli</em>. The following is the media compositions and their quantities.<br /><br />
<p><strong>Method:</strong><br/><br />
<p><br />
1.Prepare the Lysogeny Broth (LB) liquid media (1 L) as indicated below: <br/><br />
+ Bacto-Tryptone - 10 g<br/><br />
+ NaCl - 10 g<br/><br />
+ Yeast Extract - 5 g<br/><br />
Add ddH2O and 5mmol/L Tris Buffer in a measuring cylinder to ensure accuracy, to make a total of 1 liter and pH is 8.0.<br/><br />
2.Prepare the Lysogeny Broth (LB) solid media (1 L) as indicated below:<br/><br />
+ Bacto-Tryptone - 10 g<br/><br />
+ NaCl - 10 g<br/><br />
+ Yeast Extract - 5 g<br/><br />
+ Difco Agar - 15g<br/><br />
Add ddH2O and 5mmol/L Tris Buffer in a measuring cylinder to ensure accuracy, to make a total of 1 liter and pH is 8.0.<br/><br />
3. Autoclaving<br/><br />
Autoclave at 121 °C for 60 minutes. After the media cooling down enough, antibiotics Ampicillin(100mg of Ampicillin per 1ml of the media) are added. At last the media are poured 15ml on each plate and become solid.Store the plate at 4℃ refrigerator.<br />
</p><br />
</p><br />
<br />
<font face="Arial, Helvetica"><br />
<p>&nbsp;</p><br />
<br><br />
<a name="Bacterial"></a></font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Bacterial Transformation</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Transformation is commonly used to introduce recombinant plasmid DNA into bacterial strains which can transform naturally or can be made competitive for transformation by artificial means. The purpose of this technique is to introduce a recombinant plasmid DNA into a bacterial strains and to use bacteria strains to amplify the plasmid mutant-pSB1A3 for further plasmid construction.</p><br />
<p><br />
<strong>Method</strong><br /><br />
1. Take out an appropriate number of tubes that contain competent cells(100μl ) from the freezer. Immediately place the tubes on ice, so that all but the cap is surrounded by ice. Allow the cells to thaw on ice for 2-5 min.<br /><br />
2. Visually check the cells to see whether they have thawed and gently flick the cells 1-2 times to evenly resuspend the cells.<br /><br />
3. Add 10μl PCR products(mini-prep purified) to the competent cells DH-5α. Stir gently to mix and return the tube to the ice, making sure that the tube is surrounded by ice except for the cap. Repeat for additional two times for the same samples.<br /><br />
4. Incubate the tubes on ice for 30 min.<br /><br />
5. Place the tubes in a 42°C water bath for exactly 90 sec; do not shake.<br /><br />
6. Place the tubes on ice for 2 min to cool down.<br /><br />
7. Add 800 l of room temperature LB medium to each tube.<br /><br />
8. Shake the tubes vigorously at 37<a name="OLE_LINK2" id="OLE_LINK2"></a><a name="OLE_LINK1" id="OLE_LINK1">°C</a> for 45-60 min.<br /><br />
9. Centrifuge the tubes at 3K RPM for 1 min. Discard the supernatant liquor and leave 100-200 μl of the mixtures.Mix the contents and spread the whole liquid on LB agar plates containing the appropriate antibiotic ampicillin for the plasmid.<br /><br />
10. Place the plates on the bench for several min to allow excess liquid to be absorbed, and then invert and incubate overnight at 37°C (12-16 h).</p><br />
<br/><br />
</p><br />
<br />
<a name="Colony"></a></font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Colony PCR for Verification</font> </b></font></h3><br />
<font face="Arial, Helvetica"><br />
<P>Colony PCR is used to identify and select cell colonies that have the correct plasmid inserted. The procedure is a way to do several PCR operations on cell colonies in parallel, to evaluate the results and select the corresponding positive cell colonies. After an overnight growth of E.coli, we can pick up several colonies from the plate and do a colony PCR verification. Besides, the colonies we choose and should also be stored, we can incubate these colonies in one plate after every colony has been marked.</p><br />
<br />
<strong>Method</strong><br /><br />
1. Prepare the sample reaction as indicated below:<br /><br />
Total: 25μl <br /><br />
+ 0.25 μl of Ex Taq polymerase (company:Takara)<br /><br />
+ 2.5 μl of 10× Taq reaction buffer<br /><br />
+ 1.0 μl of R-NPS-F*<br /><br />
+ 1.0 μl of G-SXA-R*<br /><br />
+ 1.0 μl of plasmid mutant-psb1a3<br /><br />
+ 2.0 μl of dNTP( 25mM ) <br /><br />
+ 17.25 μl of ddH2O <br /><br />
Note: The sequences of primers R-NPS-F , G-SXA-R <br /><br />
R-NPS-F 5'-TATAGCGGCCGCCTTAAGTAAGTAAGAGTATACG-3' <br /><br />
G-SXA-R 5'-TCTCCGACTTAAGGGATCCTATAAACGCAG-3'<br /><br />
<br />
<p> 2. Set parameters for PCR to amplify desired products. </p><br />
<br />
<table><br />
<tr><br />
<td>Temperature</td> <td>Time</td> <td>Cycle</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>5min</td> <td>1</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>55˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>72˚C</td> <td>1min 20sec</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>4˚C</td> <td>7hrs</td> <td>1</td><br />
</tr><br />
</table><br />
<br />
3. Electrophorese the total system and observe the lane separation. </P><br />
<br><br />
<a name="Culture"></a></font><br />
<br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Cultivate the Bacteria</b></font></h3><br />
<font face="Arial, Helvetica"><p>According to the results of the PCR detection, positive colonies are chosen and transferred them to 5ml LB liquid media ( 5μl of ampicillin added) stored in 12.5ml centrifuge tubes. Put the centrifuge tubes in 37℃ gas bath overnight. </p><br />
<br><br />
<a name="Plasmid"></a><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Plasmid DNA Isolation</b></font></h3><br />
<p>Use E.Z.N.A.TM Plasmid Mini I to realize plasmid DNA isolation.<br /><br />
<p><strong>Method</strong><br/><br />
<br />
<ol><br />
<li>Transfer 5 ml of overnight culture into a 1.5-ml eppendorf tube labeled with group number. </li><br />
<li>Centrifuge the sample at max. speed of desk top centrifuge and RT for 1min to pellet the cells.</li><br />
<li>Discard the supernatant. Remove as much of the supernatant as possible without disturbing the cell pellet. </li><br />
<li>Repeat step 1 and 2 twice. </li><br />
<li>Resuspend the pellet completely in 250 ml of Solution I (containing RNase A) by vortexing the samples vigorously . No clumps should be visible in the tube. </li><br />
<li>Add 250 ml of Solution II and mix the sample by gently inverting the tube 4 to 6 times. Do not vortex or shake the sample vigorously. The bacterial suspension should begin to clear which have lysed the bacterial cells in this step. <strong>Warning: </strong>Do not stop here for more than five min, as the high pH hurts your DNA! </li><br />
<li>Add 350 ml of Solution III and mix by gently inverting the tube 4 to 6 times until a flocculent white precipitate forms. Do not shake vigorously, as it might break the genomic DNA. </li><br />
<li>Centrifuge at maximum speed for 10 min at room temperature to pellet the cell debris. You should see a white precipitate in the tube after the centrifugation. </li><br />
<li>While the samples are centrifuging, for each sample, label a clean HiBind Miniprep Column which is to assembled in a 2-ml collection tube</li><br />
<li>Apply the supernatants from step 8 to the columns. </li><br />
<li>Centrifuge at maximum speed for 1 min at RT. Discard the flow-through in the collection tube. </li><br />
<li>Add 500 ml of Buffer HB to wash the Hibind Miniprep Column. Centrifuge at maximum speed for 1 min at RT. Discard the flow-through in the collection tube. </li><br />
<li>Wash the column by adding 700 ml of DNA Wash Buffer diluted with absolute ethanol. Centrifuge at maximum speed for 1 min at room temperature and discard the flow-through.</li><br />
<li>Then centrifuge the tubes again for 2 min to remove all the moisture.</li><br />
<li>Place the column in a clean 1.5 ml eppendorf tube that is labeled with the plasmid name and group number. To elute the DNA, add 50 ml of Elution Buffer to the center of each column. Let the samples stand for 2 or more minutes at RT, and then centrifuge for 1 min. The sample in the centrifuge tube (bottom) is your plasmid DNA. </li><br />
<li>Discard the column and save the sample in the eppendorf tube by placing it in the freezer (-20°C). </li><br />
</ol><br />
</p><br />
<br />
<a name="#Restriction_2" ></a> <br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Restriction Enzyme Digestion and Electrophoresis</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Because the colony PCR test is so sensitive and affect markedly by environment factors. So we do a restriction enzyme digestion to ensure that the isolated plasmid is the site-directed mutated plasmid.<br /><br />
<strong>Method</strong><br /><br />
1. Prepare the control reaction as indicated below:<br /><br />
Total: 10μl<br /><br />
+ 0.5μl of Pst I restriction enzyme(company :Takara)<br /><br />
+ 1μl of 10XH buffer<br /><br />
+ 1μl of plasmid DNA<br /><br />
+ 7.5μl of ddH2O <br /><br />
2. Prepare the sample reaction as indicated below:<br /><br />
Total: 10μl<br /><br />
+ 0.5μl of Afl II restriction enzyme, (company :Takara)<br /><br />
+ 1μl of 10XM buffer<br /><br />
+ 1μl of plasmid DNA<br /><br />
+ 1.0μl of 0.01% BSA<br /><br />
+ 6.5μl of ddH2O <br /><br />
3. Electrophorese the total system and observe the lane separation.</p><br />
<br/><br />
<br />
<a name="Amplify"></a> <br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Polymerase Chain Reaction(GFP &amp; RFP) and Electrophoresis</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>GFP and RFP DNA fragments are the insert which need to be ligate to the plasmid mutant-psb1a3. Do a PCR amplification can get enough quantities for the following reactions.<br /><br />
<strong>Method </strong><br /><br />
1.Prepare the sample reaction as indicated below:<br /><br />
Total: 100μl ( PCR <a name="OLE_LINK37" id="OLE_LINK37"></a><a name="OLE_LINK36" id="OLE_LINK36">amplification</a> of GFP fragments) <br /><br />
+ 1.0μl of Taq DNA polymerase,#EP0402<br /><br />
+ 10μl of 10XTaq buffer <br /><br />
+ 10μl of MgCl2(25mM)<br /><br />
+ 10μl of dNTP(2mM)<br /><br />
+ 2μl of G-SXA-R* <br /><br />
+ 2μl of G-SXA-F*<br /><br />
+ 4μl of DNA template<br /><br />
+ 61μl of ddH2O <br /><br />
Total: 100μl(PCR amplification of RFP fragments) <br /><br />
+ 1.0μl of Taq DNA polymerase, EP0402<br /><br />
+ 10μl of 10XTaq buffer <br /><br />
+ 10μl of MgCl2(25mM)<br /><br />
+ 10μl of dNTP(2mM)<br /><br />
+ 2μl of R-NPS-R* <br /><br />
+ 2μl of R-NPS-F*<br /><br />
+ 4μl of DNA template<br /><br />
+ 61μl of ddH2O <br /><br />
<br />
Note: The sequences of primers R-NPS-F , R-NPS-R , G-SXA-F ,G-SXA-R <br /><br />
R-NPS-F 5'-TATAGCGGCCGCCTTAAGTAAGTAAGAGTATACG-3' <br /><br />
R-NPS-R 5'-CGGAGACTAGTCTGCAGATCACATAAGTAAAGTGATAATC-3' <br /><br />
G-SXA-F 5'-CTAGACTAGTTCTAGAGGCGGACTCACTATAGA-3' <br /><br />
G-SXA-R 5'-TCTCCGACTTAAGGGATCCTATAAACGCAG-3'<br /><br />
<br />
<p> 2. Set parameters for PCR to amplify desired products. </p><br />
<br />
<table><br />
<tr><br />
<td>Temperature</td> <td>Time</td> <td>Cycle</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>5min</td> <td>1</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>55˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>72˚C</td> <td>1min 20sec</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>4˚C</td> <td>7hrs</td> <td>1</td><br />
</tr><br />
</table><br />
</p><br />
</font><br />
<ul><br />
<li>Use DNA Gel Extraction Kit to purify the GFP and RFP DNA fragments after the Electrophoresis.</li><br />
</ul><br />
<p align="left"><strong>Figure</strong></p><br />
<p><font face="Arial, Helvetica"><img src="https://static.igem.org/mediawiki/2012/7/72/111.png" alt="" class="img_fl img_border" align="left"/> </font></p><br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<p>(Figure 2 : This figure shows that The PCR reaction system can amplify large quantities of GFP and RFP DNA fragments. The digestion based on the GFP and RFP DNA fragments can be done to prepare for the ligation. Lane 1 represents the template E.coli 817 can amplify the GFP and RFP DNA fragments , lane 2 represents the template E.coli 817(355.5) can also amplify the GFP and RFP DNA fragments.)<br/><br/><br />
<font face="Arial, Helvetica"><br/><br />
<a name="Restriction_Enzyme"></a><br />
</font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Double Restriction Enzyme Digestion and Electrophoresis.</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Use specific restriction enzymes to digest plasmid mutant-psb1a3,GFP and RFP to get sticky ends and purify the DNA fragment after the Electrophoresis.<br /></p><br />
<p> <strong>Method:</strong><br/><br />
</font><br />
<ul><br />
<li> Digestion of plasmid mutant-psb1a3 </li><br />
</ul><br />
<p>Prepare the sample reaction as indicated below:<br /><br />
Total: 50μl <br /><br />
+ 3.0μl of Not I restriction enzyme, #ER0591<br /><br />
+ 3.0μl of Afl II restriction enzyme, #ER0831<br /><br />
+ 5.0μl of 10X buffer O <br /><br />
+ 1.0μl of mutant-psb1a3 plasmid <br /><br />
+ 37.0μl of ddH2O<br /><br />
2. Digestion of PCR product GFP<br /><br />
Prepare the sample reaction as indicated below:<br /><br />
Total: 50μl <br /><br />
+ 3.0μl of Not I restriction enzyme, #ER0591<br /><br />
+ 3.0μl of Spe I restriction enzyme, #ER1251 <br /><br />
+ 5μl of 10X buffer Tango<br /><br />
+ 10μl of PCR products GFP<br /><br />
+ 29μl of ddH2O<br /><br />
3. Digestion of PCR product RFP<br /><br />
Prepare the sample reaction as indicated below:<br /><br />
Total: 50μl <br /><br />
+ 3.0μl of Afl II restriction enzyme, #ER0831<br /><br />
+ 3.0μl of Spe I restriction enzyme, #ER1251 <br /><br />
+ 5μl of 10X buffer Tango<br /><br />
+ 10μl of PCR products RFP<br /><br />
+ 29μl of ddH2O<br /><br />
4. Put the tubes in 37℃ environment for 4-8 hours <br /><br />
5. Use DNA Gel Extraction Kit to purify the mutant-psb1a3 fragment, GFP and RFP after digestion and named them by mutant-psb1a3 (NA) ,GFP(NS) and RFP(AS) after the Electrophoresis.</p><br />
</p><br />
<br />
<font face="Arial, Helvetica"><br><br />
<a name="Ligayion"></a> </font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Ligation</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Ligation is the process that target DNA gene is inserted into a plasmid. Both the vector and insert are prepared to have the sticky ends. These two kinds of DNA pieces are placed in a reaction tube and the proper DNA ligase, buffer, and cofactors are added for the reaction to take place. When done properly, the ligation will result in a successful combination of the insert and plasmid into one plasmid.Based on the digestion of mutant-psb1a3,GFP and RFP DNA fragments,also ligation can be done. We ligate mutant-psb1a3 vector and sticky GFP and RFP DNA fragments to construct an new plasmid mutant-psb1a3-GR. <br /><br />
1. Prepare the control reaction as indicated below:<br /><br />
Total: 10μl <br /><br />
+ 1.0μl of plasmid mutant-psb1a3 (NA) <br /><br />
+ 3.0μl of GFP(NS) <br /><br />
+ 3.0μl of RFP(AS) <br /><br />
+ 2.0μl of T4 DNA Ligase,#EL0011 <br /><br />
+ 1.0μl of 10XT4 Ligase buffer<br /><br />
Note: GFP(NS) means the product of GFP DNA fragments digested by restriction enzyme Not I and Spe I. <br /><br />
2. Prepare the sample reaction as indicated below:<br /><br />
Total: 10μl <br /><br />
+ 1.0μl of plasmid mutant-psb1a3 (NA) <br /><br />
+ 2.0μl of T4 DNA Ligase, #EL0011 <br /><br />
+ 1.0μl of 10XT4 Ligase buffer<br /><br />
+ 6.0μl of ddH2O<br /><br />
3. Put the tubes in 22℃ water bath, react for 8-12 hours. <br><br />
<a name="Bacterial_Transformation"></a> </font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Bacterial Transformation</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Transform the ligation products into the DH-5α competent cells, put the plate on 37℃ gas bath for 12-16 hours. </p><br />
<br><br />
<a name="Bacterial_Colony"></a> </font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Bacterial Colony PCR</font></b> </font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Colony PCR is used to identify and select cell colonies that have the correct plasmid insert. This procedure is a way to do several PCR operations on cell colonies in parallel, to evaluate the results and select the corresponding good cell colonies. After an overnight growth of E.coli, we can pick up some colonies from the plate and do a colony PCR verification. Besides, the colonies we choose and should also be stored, we can incubate these colonies in one plate after every colony has been marked.<br /><br />
<strong>Method</strong><br /><br />
1. Prepare the sample reaction as indicated below:<br /><br />
Total: 20μl <br /><br />
+ 0.25 μl of Ex Taq polymerase,#EP0402 <br /><br />
+ 2.0 μl of 10× Taq reaction buffer<br /><br />
+ 1.0 μl of R-NPS-F* <br /><br />
+ 1.0 μl of G-SXA-R*<br /><br />
+ 5.0 μl of bacterial colony<br /><br />
+ 2.0 μl of dNTP( 25mM ) <br /><br />
+ 8.75 μl of ddH2O <br /><br />
Note: The sequences of primers R-NPS-F , R-NPS-R , G-SXA-F ,G-SXA-R <br /><br />
R-NPS-F 5'-TATAGCGGCCGCCTTAAGTAAGTAAGAGTATACG-3' <br /><br />
R-NPS-R 5'-CGGAGACTAGTCTGCAGATCACATAAGTAAAGTGATAATC-3' <br /><br />
G-SXA-F 5'-CTAGACTAGTTCTAGAGGCGGACTCACTATAGA-3' <br /><br />
G-SXA-R 5'-TCTCCGACTTAAGGGATCCTATAAACGCAG-3'<br /><br />
<br />
<p> 2. Set parameters for PCR to amplify desired products. </p> <br />
<table><br />
<tr><br />
<td>Temperature</td> <td>Time</td> <td>Cycle</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>13.5min</td> <td>1</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>55˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>72˚C</td> <td>1min 20sec</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>4˚C</td> <td>7hrs</td> <td>1</td><br />
</tr><br />
</table><br />
<br />
</font><br />
<ul><br />
<li> Electrophorese the total system and observe the lane separation. </li><br />
</ul><br />
<p><strong>Figure</strong></p><br />
<p> <img src="https://static.igem.org/mediawiki/2012/0/07/1111.png" alt="" class="img_fl img_border" align="left"/> </p><br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<p>(Figure 3: The lane on the figure are 2k DNA fragments, it shows the GFP and RFP DNA fragments are ligated to the vectors which were isolated from the bacterial colonies.) </p><br />
<br />
<p><font face="Arial, Helvetica"><br><br />
<a name="Culture_the"></a><br />
</font></p><br />
<font face="Arial, Helvetica"><br />
</font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Cultivate the Bacteria</b></font></h3><br />
<font face="Arial, Helvetica"><p>According to the results of the PCR detection, we choose positive colonies and transfer them to 5ml LB liquid media ( 5μl of ampicillin has added) stored in 12.5ml centrifuge tubes. Put the centrifuge tubes in 37℃ gas bath overnight. </p><br />
<br />
<p><font face="Arial, Helvetica"><a name="Plasmid_DNA"></a> </font></p><br />
<font face="Arial, Helvetica"> </font> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Plasmid DNA Isolation</b></font></h3><br />
<p><font face="Arial, Helvetica">Use E.Z.N.A.TM Plasmid Mini I to isolate the constructed plasmid mutant-psb1a3-GR. </font></p><br />
<br />
<p><font face="Arial, Helvetica"><a name="Restriction_Enzyme" ></a> </font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Restriction Enzyme Digestion and Electrophoresis</b></font></h3><br />
<p>From the last step, we got the certain quantities of isolated plasmids. In this step, we do two restriction enzyme digestion reactions, one to prove that the plasmid is construct correctly ( mutant-psb1a3-GR ), one to get sticky ends preparing for the ligation.<br /><br />
<strong>Method</strong></p><br />
<p>1.Restriction Enzyme Digestion to prove that plasmid is constructed correctly</p><br />
<br />
<p align="left">1.1. Prepare the sample reaction as indicated below:<br /><br />
Total: 10μl<br /><br />
+ 1.0μl of Not I restriction enzyme,#ER0591<br /><br />
+ 1.0μl of Spe I restriction enzyme,#ER1251 <br /><br />
+ 2.0μl of Buffer Tango( 10X )<br /><br />
+ 1.5μl of plasmid mutant-psb1a3-GR<br /><br />
+ 14.5μl of ddH2O <br /><br />
1.2. Electrophorese the total system and observe the lane separation. </p><br />
<br />
<p>2. Restriction Enzyme Digestion to get sticky ends preparing for the ligation.</p><br />
<br />
<p align="left">2.1. Prepare the sample reaction as indicated below:<br /><br />
Total: 50μl<br /><br />
+ 5.0μl of Pst I restriction enzyme, #ER0611<br /><br />
+ 5.0μl of Xba I restriction enzyme, #ER0681 <br /><br />
+ 3.0μl of Buffer Tango( 10X )<br /><br />
+ 5.0μl of plasmid mutant-psb1a3-GR<br /><br />
+ 32.0μl of ddH2O <br /><br />
2.2. Electrophorese the total system and observe the lane separation.<br /><br />
2.3. Cut the gel of specific position and collect it in tubes that have measured weight. <br /><br />
2.4. Use DNA Gel Extraction Ki to purify the plasmid DNA mutant-psb1a3-GR(PX) .<br /><br />
Note: Mutant-psb1a3-GR(PX) means plasmid Mutant-psb1a3-GR digested by Pst I and Xba I.</p><br />
<br />
<p><strong>Figure</strong></p><br />
<p align="left"><strong><img src="https://static.igem.org/mediawiki/2012/d/d8/11111.png" alt="" class="img_fl img_border" align="left" /></strong></p><br />
<p>(Figure 4 : The double digestion of mutant-psb1a3 forms a linear DNA fragments and it runs slower than circle DNA fragments. This suggest that the double digestion of mutant-psb1a3 works in a high efficiency, and desired sticky ends are formed.1,3,5 are plasmids digested by restriction enzyme Pst I and Xba I from different colonies, 2,5,6 are pure plasmid mutant-psb1a3-GR, 7 is the plasmid mutant-psb1a3. )</p><br />
<p align="left">&nbsp;</p><br />
<p><font face="Arial, Helvetica"><a name="Ligation1" ></a> </font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Ligation</b></font></h3><br />
<p>Ligation is the process that target DNA gene is inserted into a plasmid. Both the vector and insert are prepared to have the sticky ends. These two kinds of DNA pieces are placed in a reaction tube and the proper DNA ligase, buffer, and cofactors are added for the reaction to take place. When done properly, the ligation will result in a successful combination of the insert and plasmid into one plasmid.Based on the digestion of mutant-psb1a3-GR, terminator DNA fragments,ligation can be done. We ligate mutant-psb1a3-GR vector and sticky terminator DNA fragments to construct an new plasmid mutant-psb1a3-GR-t.By detecting the quantities of GFP and RFP, terminator efficiency can be calculated. <br /><br />
1. Prepare the control reaction as indicated below:<br /><br />
Total: 10μl <br /><br />
+ 1.0μl of plasmid mutant-psb1a3 (NA) <br /><br />
+ 6.0μl of terminator<br /><br />
+ 2.0μl of T4 DNA Ligase , #EL0011 <br /><br />
+ 1.0μl of 10XT4 Ligase buffer<br /><br />
2. Put the tubes in 22℃ water bath, react for 8-12 hours. </p><br />
<p><font face="Arial, Helvetica"><a name="Bacteria" id="Culture_the"></a> </font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Bacteria transformation</b></font></h3><br />
<p>Transform the ligation products into the DH-5α competent cells, put the plate on 37℃ gas bath for 12-16 hours. </p><br />
<p><font face="Arial, Helvetica"><a name="Cultivate"></a> </font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Cultivate the Bacteria</b></font></h3><br />
<p>According to the results of the PCR detection, we choose positive colonies and transfer them to 5ml LB liquid media ( 5μl of ampicillin has added) stored in 12.5ml centrifuge tubes. Put the centrifuge tubes in 37℃ gas bath overnight. </p><br />
<br />
<p><font face="Arial, Helvetica"><a name="Flow" ></a> </font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Flow Cytometer Analysis</b></font></h3><br />
<p>Flow cytometry is a laser based, biophysical technology employed in cell counting, sorting, biomarker detection and protein engineering, by suspending cells in a stream of fluid and passing them by an electronic detection apparatus. It allows simultaneous multiparametric analysis of the physical and/or chemical characteristics of up to thousands of particles per second. </p><br />
<p>Each fluorophore has a characteristic peak excitation and emission wavelength, and the emission spectra often overlap. Consequently, the combination of labels which can be used depends on the wavelength of the lamp(s) or laser(s) used to excite the fluorochromes and on the detectors available. It is practical that the expression of RFP and GFP can be measured at the same time.</p><br />
<p><strong>Method:</strong><br/><br />
1. Materials:<br/><br />
&nbsp;&nbsp;1.1. NaCl solution( 0.9% ,M/V)<br/><br />
&nbsp;&nbsp;1.2. 75% ethanol<br/><br />
2. Procedures:<br/><br />
&nbsp;&nbsp;2.1. Bacterium are harvested by centrifuge at 10000g for 30s, then discard the supernatant. Repeat this step again until we got adequate bacterium.<br/><br />
&nbsp;&nbsp;2.2. Suspend the bacterium With NaCl solution(0.9%) and vibrate the centrifuge tubes until the bacterium are distributed homogeneous.<br/><br />
&nbsp;&nbsp;2.3. Load the bacteria containing pSB1A3 to Flow Cytometer (Beckman), set the parameters. Measure the GFP-FL1 and RFP-FL2 by Cytometer. </p><br />
<br />
<p><font face="Arial, Helvetica"><a name="Fluorescence"></a> </font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Fluorescence Microscope</b></font></h3><br />
<p><strong>Method</strong><br /><br />
Add 10μl of former bacteria solution to micro slide and cover with coverslip.Then placed it on the Fluorescence Microscope with 488nm light activating and observe the GFP and RFP. </p><br />
<br> <br />
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</html></div>M.B.ZHOUhttp://2012.igem.org/Team:SUSTC-Shenzhen-B/protocol1Team:SUSTC-Shenzhen-B/protocol12012-10-26T20:38:21Z<p>M.B.ZHOU: </p>
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.pp_arrow_previous:hover{opacity:.7}div.pp_default .pp_description{font-size:11px;font-weight:700;line-height:14px;margin:5px 50px 5px 0}div.pp_default .pp_bottom .pp_left{background:url(../images/default/sprite.png) -78px -127px no-repeat}div.pp_default .pp_bottom .pp_middle{background:url(../images/default/sprite_x.png) bottom left repeat-x}div.pp_default .pp_bottom .pp_right{background:url(../images/default/sprite.png) -112px -127px no-repeat}div.pp_default .pp_loaderIcon{background:url(../images/default/loader.gif) center center no-repeat}div.light_rounded .pp_top .pp_left{background:url(../images/light_rounded/sprite.png) -88px -53px no-repeat}div.light_rounded .pp_top .pp_right{background:url(../images/light_rounded/sprite.png) -110px -53px no-repeat}div.light_rounded .pp_next:hover{background:url(../images/light_rounded/btnNext.png) center right no-repeat;cursor:pointer}div.light_rounded .pp_previous:hover{background:url(../images/light_rounded/btnPrevious.png) center left no-repeat;cursor:pointer}div.light_rounded .pp_expand{background:url(../images/light_rounded/sprite.png) -31px -26px no-repeat;cursor:pointer}div.light_rounded .pp_expand:hover{background:url(../images/light_rounded/sprite.png) -31px -47px no-repeat;cursor:pointer}div.light_rounded .pp_contract{background:url(../images/light_rounded/sprite.png) 0 -26px no-repeat;cursor:pointer}div.light_rounded .pp_contract:hover{background:url(../images/light_rounded/sprite.png) 0 -47px no-repeat;cursor:pointer}div.light_rounded .pp_close{background:url(../images/light_rounded/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:75px}div.light_rounded .pp_nav .pp_play{background:url(../images/light_rounded/sprite.png) -1px -100px no-repeat;height:15px;width:14px}div.light_rounded .pp_nav .pp_pause{background:url(../images/light_rounded/sprite.png) -24px -100px no-repeat;height:15px;width:14px}div.light_rounded .pp_arrow_previous{background:url(../images/light_rounded/sprite.png) 0 -71px no-repeat}div.light_rounded .pp_arrow_next{background:url(../images/light_rounded/sprite.png) -22px -71px no-repeat}div.light_rounded .pp_bottom .pp_left{background:url(../images/light_rounded/sprite.png) -88px -80px no-repeat}div.light_rounded .pp_bottom .pp_right{background:url(../images/light_rounded/sprite.png) -110px -80px no-repeat}div.dark_rounded .pp_top .pp_left{background:url(../images/dark_rounded/sprite.png) -88px -53px no-repeat}div.dark_rounded .pp_top .pp_right{background:url(../images/dark_rounded/sprite.png) -110px -53px no-repeat}div.dark_rounded .pp_content_container .pp_left{background:url(../images/dark_rounded/contentPattern.png) top left repeat-y}div.dark_rounded .pp_content_container .pp_right{background:url(../images/dark_rounded/contentPattern.png) top right repeat-y}div.dark_rounded .pp_next:hover{background:url(../images/dark_rounded/btnNext.png) center right no-repeat;cursor:pointer}div.dark_rounded .pp_previous:hover{background:url(../images/dark_rounded/btnPrevious.png) center left no-repeat;cursor:pointer}div.dark_rounded .pp_expand{background:url(../images/dark_rounded/sprite.png) -31px -26px no-repeat;cursor:pointer}div.dark_rounded .pp_expand:hover{background:url(../images/dark_rounded/sprite.png) -31px -47px no-repeat;cursor:pointer}div.dark_rounded .pp_contract{background:url(../images/dark_rounded/sprite.png) 0 -26px no-repeat;cursor:pointer}div.dark_rounded .pp_contract:hover{background:url(../images/dark_rounded/sprite.png) 0 -47px no-repeat;cursor:pointer}div.dark_rounded .pp_close{background:url(../images/dark_rounded/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:75px}div.dark_rounded .pp_description{color:#fff;margin-right:85px}div.dark_rounded .pp_nav .pp_play{background:url(../images/dark_rounded/sprite.png) -1px -100px no-repeat;height:15px;width:14px}div.dark_rounded .pp_nav .pp_pause{background:url(../images/dark_rounded/sprite.png) -24px -100px no-repeat;height:15px;width:14px}div.dark_rounded .pp_arrow_previous{background:url(../images/dark_rounded/sprite.png) 0 -71px no-repeat}div.dark_rounded .pp_arrow_next{background:url(../images/dark_rounded/sprite.png) -22px -71px no-repeat}div.dark_rounded .pp_bottom .pp_left{background:url(../images/dark_rounded/sprite.png) -88px -80px no-repeat}div.dark_rounded .pp_bottom .pp_right{background:url(../images/dark_rounded/sprite.png) -110px -80px no-repeat}div.dark_rounded .pp_loaderIcon{background:url(../images/dark_rounded/loader.gif) center center no-repeat}div.dark_square .pp_left,div.dark_square .pp_middle,div.dark_square .pp_right,div.dark_square .pp_content{background:#000}div.dark_square .pp_description{color:#fff;margin:0 85px 0 0}div.dark_square .pp_loaderIcon{background:url(../images/dark_square/loader.gif) center center no-repeat}div.dark_square .pp_expand{background:url(../images/dark_square/sprite.png) -31px -26px no-repeat;cursor:pointer}div.dark_square .pp_expand:hover{background:url(../images/dark_square/sprite.png) -31px -47px no-repeat;cursor:pointer}div.dark_square .pp_contract{background:url(../images/dark_square/sprite.png) 0 -26px no-repeat;cursor:pointer}div.dark_square .pp_contract:hover{background:url(../images/dark_square/sprite.png) 0 -47px no-repeat;cursor:pointer}div.dark_square .pp_close{background:url(../images/dark_square/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:75px}div.dark_square .pp_nav{clear:none}div.dark_square .pp_nav .pp_play{background:url(../images/dark_square/sprite.png) -1px -100px no-repeat;height:15px;width:14px}div.dark_square .pp_nav .pp_pause{background:url(../images/dark_square/sprite.png) -24px -100px no-repeat;height:15px;width:14px}div.dark_square .pp_arrow_previous{background:url(../images/dark_square/sprite.png) 0 -71px no-repeat}div.dark_square .pp_arrow_next{background:url(../images/dark_square/sprite.png) -22px -71px no-repeat}div.dark_square .pp_next:hover{background:url(../images/dark_square/btnNext.png) center right no-repeat;cursor:pointer}div.dark_square .pp_previous:hover{background:url(../images/dark_square/btnPrevious.png) center left no-repeat;cursor:pointer}div.light_square .pp_expand{background:url(../images/light_square/sprite.png) -31px -26px no-repeat;cursor:pointer}div.light_square .pp_expand:hover{background:url(../images/light_square/sprite.png) -31px -47px no-repeat;cursor:pointer}div.light_square .pp_contract{background:url(../images/light_square/sprite.png) 0 -26px no-repeat;cursor:pointer}div.light_square .pp_contract:hover{background:url(../images/light_square/sprite.png) 0 -47px no-repeat;cursor:pointer}div.light_square .pp_close{background:url(../images/light_square/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:75px}div.light_square .pp_nav .pp_play{background:url(../images/light_square/sprite.png) -1px -100px no-repeat;height:15px;width:14px}div.light_square .pp_nav .pp_pause{background:url(../images/light_square/sprite.png) -24px -100px no-repeat;height:15px;width:14px}div.light_square .pp_arrow_previous{background:url(../images/light_square/sprite.png) 0 -71px no-repeat}div.light_square .pp_arrow_next{background:url(../images/light_square/sprite.png) -22px -71px no-repeat}div.light_square .pp_next:hover{background:url(../images/light_square/btnNext.png) center right no-repeat;cursor:pointer}div.light_square .pp_previous:hover{background:url(../images/light_square/btnPrevious.png) center left no-repeat;cursor:pointer}div.facebook .pp_top .pp_left{background:url(../images/facebook/sprite.png) -88px -53px no-repeat}div.facebook .pp_top .pp_middle{background:url(../images/facebook/contentPatternTop.png) top left repeat-x}div.facebook .pp_top .pp_right{background:url(../images/facebook/sprite.png) -110px -53px no-repeat}div.facebook .pp_content_container .pp_left{background:url(../images/facebook/contentPatternLeft.png) top left repeat-y}div.facebook .pp_content_container .pp_right{background:url(../images/facebook/contentPatternRight.png) top right repeat-y}div.facebook .pp_expand{background:url(../images/facebook/sprite.png) -31px -26px no-repeat;cursor:pointer}div.facebook .pp_expand:hover{background:url(../images/facebook/sprite.png) -31px -47px no-repeat;cursor:pointer}div.facebook .pp_contract{background:url(../images/facebook/sprite.png) 0 -26px no-repeat;cursor:pointer}div.facebook .pp_contract:hover{background:url(../images/facebook/sprite.png) 0 -47px no-repeat;cursor:pointer}div.facebook .pp_close{background:url(../images/facebook/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:22px}div.facebook .pp_description{margin:0 37px 0 0}div.facebook .pp_loaderIcon{background:url(../images/facebook/loader.gif) center center no-repeat}div.facebook .pp_arrow_previous{background:url(../images/facebook/sprite.png) 0 -71px no-repeat;height:22px;margin-top:0;width:22px}div.facebook .pp_arrow_previous.disabled{background-position:0 -96px;cursor:default}div.facebook .pp_arrow_next{background:url(../images/facebook/sprite.png) -32px -71px no-repeat;height:22px;margin-top:0;width:22px}div.facebook .pp_arrow_next.disabled{background-position:-32px -96px;cursor:default}div.facebook .pp_nav{margin-top:0}div.facebook .pp_nav p{font-size:15px;padding:0 3px 0 4px}div.facebook .pp_nav .pp_play{background:url(../images/facebook/sprite.png) -1px -123px no-repeat;height:22px;width:22px}div.facebook .pp_nav .pp_pause{background:url(../images/facebook/sprite.png) -32px -123px no-repeat;height:22px;width:22px}div.facebook .pp_next:hover{background:url(../images/facebook/btnNext.png) center right no-repeat;cursor:pointer}div.facebook .pp_previous:hover{background:url(../images/facebook/btnPrevious.png) center left no-repeat;cursor:pointer}div.facebook .pp_bottom .pp_left{background:url(../images/facebook/sprite.png) -88px -80px no-repeat}div.facebook .pp_bottom .pp_middle{background:url(../images/facebook/contentPatternBottom.png) top left repeat-x}div.facebook .pp_bottom .pp_right{background:url(../images/facebook/sprite.png) -110px -80px no-repeat}div.pp_pic_holder a:focus{outline:0}div.pp_overlay{background:#000;display:none;left:0;position:absolute;top:0;width:100%;z-index:9500}div.pp_pic_holder{display:none;position:absolute;width:100px;z-index:10000}.pp_content{height:40px;min-width:40px}* html .pp_content{width:40px}.pp_content_container{position:relative;text-align:left;width:100%}.pp_content_container .pp_left{padding-left:20px}.pp_content_container .pp_right{padding-right:20px}.pp_content_container .pp_details{float:left;margin:10px 0 2px}.pp_description{display:none;margin:0}.pp_social{float:left;margin:0}.pp_social .facebook{float:left;margin-left:5px;overflow:hidden;width:55px}.pp_social .twitter{float:left}.pp_nav{clear:right;float:left;margin:3px 10px 0 0}.pp_nav p{float:left;margin:2px 4px;white-space:nowrap}.pp_nav .pp_play,.pp_nav .pp_pause{float:left;margin-right:4px;text-indent:-10000px}a.pp_arrow_previous,a.pp_arrow_next{display:block;float:left;height:15px;margin-top:3px;overflow:hidden;text-indent:-10000px;width:14px}.pp_hoverContainer{position:absolute;top:0;width:100%;z-index:2000}.pp_gallery{display:none;left:50%;margin-top:-50px;position:absolute;z-index:10000}.pp_gallery div{float:left;overflow:hidden;position:relative}.pp_gallery ul{float:left;height:35px;margin:0 0 0 5px;padding:0;position:relative;white-space:nowrap}.pp_gallery ul a{border:1px rgba(0,0,0,.5) solid;display:block;float:left;height:33px;overflow:hidden}.pp_gallery ul a img{border:0}.pp_gallery li{display:block;float:left;margin:0 5px 0 0;padding:0}.pp_gallery li.default a{background:url(../images/facebook/default_thumbnail.gif) 0 0 no-repeat;display:block;height:33px;width:50px}.pp_gallery .pp_arrow_previous,.pp_gallery .pp_arrow_next{margin-top:7px!important}a.pp_next{background:url(../images/light_rounded/btnNext.png) 10000px 10000px no-repeat;display:block;float:right;height:100%;text-indent:-10000px;width:49%}a.pp_previous{background:url(../images/light_rounded/btnNext.png) 10000px 10000px no-repeat;display:block;float:left;height:100%;text-indent:-10000px;width:49%}a.pp_expand,a.pp_contract{cursor:pointer;display:none;height:20px;position:absolute;right:30px;text-indent:-10000px;top:10px;width:20px;z-index:20000}a.pp_close{display:block;line-height:22px;position:absolute;right:0;text-indent:-10000px;top:0}.pp_loaderIcon{display:block;height:24px;left:50%;margin:-12px 0 0 -12px;position:absolute;top:50%;width:24px}#pp_full_res{line-height:1!important}#pp_full_res .pp_inline{text-align:left}#pp_full_res .pp_inline p{margin:0 0 15px}div.ppt{color:#fff;display:none;font-size:17px;margin:0 0 5px 15px;z-index:9999}div.pp_default .pp_content,div.light_rounded .pp_content{background-color:#fff}div.pp_default #pp_full_res .pp_inline,div.light_rounded .pp_content .ppt,div.light_rounded #pp_full_res .pp_inline,div.light_square .pp_content .ppt,div.light_square #pp_full_res .pp_inline,div.facebook .pp_content .ppt,div.facebook #pp_full_res .pp_inline{color:#000}div.pp_default .pp_gallery ul li a:hover,div.pp_default .pp_gallery ul li.selected a,.pp_gallery ul a:hover,.pp_gallery li.selected a{border-color:#fff}div.pp_default .pp_details,div.light_rounded .pp_details,div.dark_rounded .pp_details,div.dark_square .pp_details,div.light_square .pp_details,div.facebook .pp_details{position:relative}div.light_rounded .pp_top .pp_middle,div.light_rounded .pp_content_container .pp_left,div.light_rounded .pp_content_container .pp_right,div.light_rounded .pp_bottom .pp_middle,div.light_square .pp_left,div.light_square .pp_middle,div.light_square .pp_right,div.light_square .pp_content,div.facebook .pp_content{background:#fff}div.light_rounded .pp_description,div.light_square .pp_description{margin-right:85px}div.light_rounded .pp_gallery a.pp_arrow_previous,div.light_rounded .pp_gallery a.pp_arrow_next,div.dark_rounded .pp_gallery a.pp_arrow_previous,div.dark_rounded .pp_gallery a.pp_arrow_next,div.dark_square .pp_gallery a.pp_arrow_previous,div.dark_square .pp_gallery a.pp_arrow_next,div.light_square .pp_gallery a.pp_arrow_previous,div.light_square .pp_gallery a.pp_arrow_next{margin-top:12px!important}div.light_rounded .pp_arrow_previous.disabled,div.dark_rounded .pp_arrow_previous.disabled,div.dark_square .pp_arrow_previous.disabled,div.light_square .pp_arrow_previous.disabled{background-position:0 -87px;cursor:default}div.light_rounded .pp_arrow_next.disabled,div.dark_rounded .pp_arrow_next.disabled,div.dark_square .pp_arrow_next.disabled,div.light_square .pp_arrow_next.disabled{background-position:-22px -87px;cursor:default}div.light_rounded .pp_loaderIcon,div.light_square .pp_loaderIcon{background:url(../images/light_rounded/loader.gif) center center no-repeat}div.dark_rounded .pp_top .pp_middle,div.dark_rounded .pp_content,div.dark_rounded .pp_bottom .pp_middle{background:url(../images/dark_rounded/contentPattern.png) top left repeat}div.dark_rounded .currentTextHolder,div.dark_square .currentTextHolder{color:#c4c4c4}div.dark_rounded #pp_full_res .pp_inline,div.dark_square #pp_full_res .pp_inline{color:#fff}.pp_top,.pp_bottom{height:20px;position:relative}* html .pp_top,* html .pp_bottom{padding:0 20px}.pp_top .pp_left,.pp_bottom .pp_left{height:20px;left:0;position:absolute;width:20px}.pp_top .pp_middle,.pp_bottom .pp_middle{height:20px;left:20px;position:absolute;right:20px}* html .pp_top .pp_middle,* html .pp_bottom .pp_middle{left:0;position:static}.pp_top .pp_right,.pp_bottom .pp_right{height:20px;left:auto;position:absolute;right:0;top:0;width:20px}.pp_fade,.pp_gallery li.default a img{display:none}<br />
<br />
</style><br />
<script type="text/javascript"><br />
<br />
/*<br />
* Superfish v1.4.8 - jQuery menu widget<br />
* Copyright (c) 2008 Joel Birch<br />
*<br />
* Dual licensed under the MIT and GPL licenses:<br />
* http://www.opensource.org/licenses/mit-license.php<br />
* http://www.gnu.org/licenses/gpl.html<br />
*<br />
* CHANGELOG: http://users.tpg.com.au/j_birch/plugins/superfish/changelog.txt<br />
*/<br />
<br />
;(function($){<br />
$.fn.superfish = function(op){<br />
<br />
var sf = $.fn.superfish,<br />
c = sf.c,<br />
$arrow = $(['<span class="',c.arrowClass,'"> &#187;</span>'].join('')),<br />
over = function(){<br />
var $$ = $(this), menu = getMenu($$);<br />
clearTimeout(menu.sfTimer);<br />
$$.showSuperfishUl().siblings().hideSuperfishUl();<br />
},<br />
out = function(){<br />
var $$ = $(this), menu = getMenu($$), o = sf.op;<br />
clearTimeout(menu.sfTimer);<br />
menu.sfTimer=setTimeout(function(){<br />
o.retainPath=($.inArray($$[0],o.$path)>-1);<br />
$$.hideSuperfishUl();<br />
//if (o.$path.length &amp;&amp; $$.parents(['li.',o.hoverClass].join('')).length<1){over.call(o.$path);}<br />
if (o.$path.length) {<br />
if ($$.parents(['li.',o.hoverClass].join('')).length<1){over.call(o.$path);}<br />
}<br />
},o.delay); <br />
},<br />
getMenu = function($menu){<br />
var menu = $menu.parents(['ul.',c.menuClass,':first'].join(''))[0];<br />
sf.op = sf.o[menu.serial];<br />
return menu;<br />
},<br />
addArrow = function($a){ $a.addClass(c.anchorClass).append($arrow.clone()); };<br />
<br />
return this.each(function() {<br />
var s = this.serial = sf.o.length;<br />
var o = $.extend({},sf.defaults,op);<br />
o.$path = $('li.'+o.pathClass,this).slice(0,o.pathLevels).each(function(){<br />
$(this).addClass([o.hoverClass,c.bcClass].join(' '))<br />
.filter('li:has(ul)').removeClass(o.pathClass);<br />
});<br />
sf.o[s] = sf.op = o;<br />
var bcheck = false;<br />
if ($.fn.hoverIntent) {<br />
if (!o.disableHI) bcheck = true;<br />
}<br />
<br />
$('li:has(ul)',this)[(bcheck) ? 'hoverIntent' : 'hover'](over,out).each(function() {<br />
if (o.autoArrows) addArrow( $('>a:first-child',this) );<br />
})<br />
.not('.'+c.bcClass)<br />
.hideSuperfishUl();<br />
<br />
var $a = $('a',this);<br />
$a.each(function(i){<br />
var $li = $a.eq(i).parents('li');<br />
$a.eq(i).focus(function(){over.call($li);}).blur(function(){out.call($li);});<br />
});<br />
o.onInit.call(this);<br />
<br />
}).each(function() {<br />
var menuClasses = [c.menuClass];<br />
//if (sf.op.dropShadows &amp;&amp; !($.browser.msie &amp;&amp; $.browser.version < 7)) menuClasses.push(c.shadowClass);<br />
if (sf.op.dropShadows) if (!$.browser.msie) if (!($.browser.version < 7)) menuClasses.push(c.shadowClass);<br />
$(this).addClass(menuClasses.join(' '));<br />
});<br />
};<br />
<br />
var sf = $.fn.superfish;<br />
sf.o = [];<br />
sf.op = {};<br />
sf.IE7fix = function(){<br />
var o = sf.op;<br />
//if ($.browser.msie &amp;&amp; $.browser.version > 6 &amp;&amp; o.dropShadows &amp;&amp; o.animation.opacity!=undefined)<br />
if ($.browser.msie) if($.browser.version > 6) if (o.dropShadows) if (o.animation.opacity!=undefined)<br />
this.toggleClass(sf.c.shadowClass+'-off');<br />
};<br />
sf.c = {<br />
bcClass : 'sf-breadcrumb',<br />
menuClass : 'sf-js-enabled',<br />
anchorClass : 'sf-with-ul',<br />
arrowClass : 'sf-sub-indicator',<br />
shadowClass : 'sf-shadow'<br />
};<br />
sf.defaults = {<br />
hoverClass : 'sfHover',<br />
pathClass : 'overideThisToUse',<br />
pathLevels : 1,<br />
delay : 800,<br />
animation : {opacity:'show'},<br />
speed : 'normal',<br />
autoArrows : true,<br />
dropShadows : true,<br />
disableHI : false, // true disables hoverIntent detection<br />
onInit : function(){}, // callback functions<br />
onBeforeShow: function(){},<br />
onShow : function(){},<br />
onHide : function(){}<br />
};<br />
$.fn.extend({<br />
hideSuperfishUl : function(){<br />
var o = sf.op,<br />
not = (o.retainPath===true) ? o.$path : '';<br />
o.retainPath = false;<br />
var $ul = $(['li.',o.hoverClass].join(''),this).add(this).not(not).removeClass(o.hoverClass)<br />
.find('>ul').hide().css('visibility','hidden');<br />
o.onHide.call($ul);<br />
return this;<br />
},<br />
showSuperfishUl : function(){<br />
var o = sf.op,<br />
sh = sf.c.shadowClass+'-off',<br />
$ul = this.addClass(o.hoverClass)<br />
.find('>ul:hidden').css('visibility','visible');<br />
sf.IE7fix.call($ul);<br />
o.onBeforeShow.call($ul);<br />
$ul.animate(o.animation,o.speed,function(){ sf.IE7fix.call($ul); o.onShow.call($ul); });<br />
return this;<br />
}<br />
});<br />
<br />
})(jQuery);<br />
<br />
/*<br />
* Supersubs v0.2b - jQuery plugin<br />
* Copyright (c) 2008 Joel Birch<br />
*<br />
* Dual licensed under the MIT and GPL licenses:<br />
* http://www.opensource.org/licenses/mit-license.php<br />
* http://www.gnu.org/licenses/gpl.html<br />
*<br />
*<br />
* This plugin automatically adjusts submenu widths of suckerfish-style menus to that of<br />
* their longest list item children. If you use this, please expect bugs and report them<br />
* to the jQuery Google Group with the word 'Superfish' in the subject line.<br />
*<br />
*/<br />
<br />
(function($){ // $ will refer to jQuery within this closure<br />
<br />
$.fn.supersubs = function(options){<br />
var opts = $.extend({}, $.fn.supersubs.defaults, options);<br />
// return original object to support chaining<br />
return this.each(function() {<br />
// cache selections<br />
var $$ = $(this);<br />
// support metadata<br />
var o = $.meta ? $.extend({}, opts, $$.data()) : opts;<br />
// get the font size of menu.<br />
// .css('fontSize') returns various results cross-browser, so measure an em dash instead<br />
var fontsize = $('<li id="menu-fontsize">&#8212;</li>').css({<br />
'padding' : 0,<br />
'position' : 'absolute',<br />
'top' : '-999em',<br />
'width' : 'auto'<br />
}).appendTo($$).width(); //clientWidth is faster, but was incorrect here<br />
// remove em dash<br />
$('#menu-fontsize').remove();<br />
// cache all ul elements<br />
$ULs = $$.find('ul');<br />
// loop through each ul in menu<br />
$ULs.each(function(i) { <br />
// cache this ul<br />
var $ul = $ULs.eq(i);<br />
// get all (li) children of this ul<br />
var $LIs = $ul.children();<br />
// get all anchor grand-children<br />
var $As = $LIs.children('a');<br />
// force content to one line and save current float property<br />
var liFloat = $LIs.css('white-space','nowrap').css('float');<br />
// remove width restrictions and floats so elements remain vertically stacked<br />
var emWidth = $ul.add($LIs).add($As).css({<br />
'float' : 'none',<br />
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<article><br />
<h1><p>Lab Protocol</p></h1><br />
</font><br />
<font face="Arial, Helvetica"><br />
<p><a href="#Site-Directed_Mutagenesis">1. Site-Directed Mutagenesis</a></p><br />
<p><a href="#Restriction">2. Mutation Verification by Restriction Enzyme Digestion</a></p><br />
<p><a href="#Media">3. Media Preparation</a></p><br />
<p><a href="#Bacterial">4. Bacterial Transformation</a></p><br />
<p><a href="#Colony">5. Colony PCR for Verification</a></p><br />
<p><a href="#Culture">6. Cultivate the Bacteria</a></p><br />
<p><a href="#Plasmid">7. Plasmid DNA Isolation</a></p><br />
<p><a href="#Restriction_2">8. Mutation Verification by Restriction Enzyme Digestion </a></p><br />
<p><a href="#Amplify">9. Polymerase Chain Reaction and Electrophoresis</a></p><br />
<p><a href="#Electrophoresis">10. Double Restriction Enzyme Digestion and Electrophoresis </a></p><br />
<p><a href="#Ligation">11. Ligation</a></p><br />
<p><a href="#Bacterial_Transformation">12. Bacterial Transformation</a></p><br />
<p><a href="#bacterial_colony">13. Bacterial Colony PCR</a></p><br />
<p><a href="#Culture_the">14. Cultivate the Bacteria</a></p><br />
<p><a href="#Plasmid_DNA">15. Plasmid DNA Isolation</a></p><br />
<p><a href="#Restriction_Enzyme">16. Restriction Enzyme Digestion and Electrophoresis</a></p><br />
<p><a href="#Ligation1">17. Ligation</a></p><br />
<p><a href="#Bacteria">18. Bacteria Transformation</a></p><br />
<p><a href="#Cultivate">19. Cultivate the Bacteria</a></p><br />
<p><a href="#Flow">20. Flow Cytometer Analysis</a></p><br />
<p><a href="#Fluorescence">21. Fluorescence Microscope </a></p><br />
<br><br />
<a name="Site-Directed_Mutagenesis" ></a></font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Site-Directed Mutagenesis</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Plasmid pSB1A3 was chosen as a backbone vector for cloning. The Pst I site in pSB1A3 was mutated to Afl II site to facilitate following cloning processes. Proper primers were designed and PCR-based site-directed mutageneis were carried out to generate this mutation as descbibed below. </p><br />
<br />
<p><strong>Method:</strong></p><br />
<br />
<p>1. Set up PCR assay tubes as described below:<br />
<br/><br />
Total: 25 μl<br /><br />
+ 0.25 μl of Ex Taq polymerase <br /><br />
+ 2.5 μl of 10× Taq reaction buffer<br /><br />
+ 2.0 μl of dNTP(2 mM) <br /><br />
+ 1.0 μl of template (E.coli plasmid 817) <br /><br />
+ 1.0 μl of oligonucleotide primer PtoA-F*<br /><br />
+ 1.0 μl of oligonucleotide primer PtoA-R* <br /><br />
+ 18.25 μl of ddH2O <br /><br />
*The sequences of primer pair PtoA-F and PtoA-R.<br /><br />
PtoA-F 5'-CCACCTGACGTCTAAGAAAC-3'<br /><br />
PtoA-R 5'-ATGATCATCGCCGGCGAATTCAGGC-3' <br /><br />
</p><br />
<p> 2. Set parameters for PCR to amplify desired products. </p><br />
<br />
<table><br />
<tr><br />
<td>Temperature</td> <td>Time</td> <td>Cycle</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>5min</td> <td>1</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>55˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>72˚C</td> <td>1min 20sec</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>4˚C</td> <td>7hrs</td> <td>1</td><br />
</tr><br />
</table><br />
<br />
<font face="Arial, Helvetica"><br />
<p><br><br />
<a name="Restriction" ></a> </p><br />
</font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Restriction Enzyme Digestion for Verification</font></b></font></h3><br />
<p align="left">To verify whether PstI site in pSB1A3 was successfully mutated to AflII site, we performed restriction enzyme digestion experiments. </p><br />
<p>Bacterial plasmids are double-stranded circular DNA molecules and uncut plasmid DNA can be in any of three forms - nicked circular, linear, closed supercoiled. When run on an agarose gel one frequently will see these forms as different bands with closed supercoiled form migrates the fastest, linear form migrates the slowest, and nicked circular migrates in between. If the PStI site was successfully mutated to AflII site, we expected to see increased linear form of pSB1A3 when digested with AflII. SpeI-cut pSB1A3 was served as a positive control. </p><br /><br />
<strong>Method</strong><br /><br />
1. Set up Pst I digestion in 1.5 ml eppendorf tubes as described below:<br /><br />
Total: 10μl<br /><br />
+ 0.5μl of Pst I restriction enzyme (company :Takara)<br /><br />
+ 1μl of 10XH buffer<br /><br />
+ 1μl of plasmid DNA<br /><br />
+ 7.5μl of ddH2O <br /><br />
2. Set up Afl II digestion in 1.5 ml eppendorf tubes as described below:<br /><br />
Total: 10μl<br /><br />
+ 0.5μl of Afl II restriction enzyme, (company :Takara)<br /><br />
+ 1μl of 10XM buffer<br /><br />
+ 1μl of plasmid DNA<br /><br />
+ 1.0μl of 0.01% BSA<br /><br />
+ 6.5μl of ddH2O<br /><br />
3. Incubate the eppendorf tubes in 37℃ water bath for 1-2 hr. <br/><br />
4. Prepare 1% agarose gel in Conical flask. Weigh 0.6 g agarose and add 60 ml 1x TAE (diluted from 50x TAE). Cover the Conical flask with silver paper to avoid the loss of water vapor. Place the Conical flask in the microwave and microwave for 1 minute with a middle power. Take it out and shake gently till the solution is homogeneous,(BE CAREFUL to watch the solution closely when shake it–it superheats and can boil over and cause severe burns). Continue microwave and swirl until solution is seen clear and homogeneous with no existence of solid. After cool down the agarose gel briefly, add 3 μl of Gelred (10000x ) and mix well. Pour the agarose gel in gel casting apparatus and insert combs.<br/><br />
5. By inserting the pipette tip below the TAE liquid and into the well, add 5 μl of 1kb DNA ladder solution to first (and last if desired) well, skip one well, then begin adding the 5μl of digested DNA solutions mixed with 1 μl loading buffer (6x) to the wells. <br/><br />
6. Place the cover on the electrophoresis unit, plug into the power source, and turn on voltage to 120V, set time to 30 minutes, and press the start button twice,until the bubbles are seen. DNA separation can be observed as time goes on by turning off the power supply then gently removing the basin from the electrophoresis unit (be careful not to let the gel slip out of the basin) and placing on the UV transilluminator to see DNA bands. <br/><br />
7. When the desired level of separation is obtained, the basin can be placed on the transilluminator for picture taking(Of the absence of transilluminator,we use camera to take pictures with the UV light ). <br/><br />
8. Cut the gel of specific position and collect it in tubes that have measured weight. <br/><br />
9. Use DNA Gel Extraction Ki to purify the plasmid DNA mutant-psb1a3.<br />
RPF (SpeI/AflII-digested), GFP (SpeI/AflII-digested) fragments were ligated into this vector, which was followed by insertion of designed terminator sequences between RFP and GFP, respectively.</p><br />
<font face="Arial, Helvetica"><br />
<p> <img src="https://static.igem.org/mediawiki/2012/5/59/11.png" alt="" class="img_fl img_border" align="left"/> </p><br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<p>(Figure 1 : This figure shows that the site-directed mutagenesis succeed ,we successfully change a restriction enzyme cutting site named Pst I to Afl II. Lane 1 represents the plasmid mutant-psb1a3, lane 2 shows that the mutant-psb1a3 cannot be digested by restriction enzyme Spe I ,lane 3 shows that mutant-psb1a3 can be digested by restriction enzyme Afl II.) </p><br />
<br />
<br />
<a name="Media"></a></font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Media Preparation</font></b></font></h3><br />
<p align="left">For all experiments involving the bacterial biomass and experimentation, proper media is chosen to grow the cells. Commonly,we use Lysogeny broth media for <em>E. coli</em>. The following is the media compositions and their quantities.<br /><br />
<p><strong>Method:</strong></p><br />
<p><br />
1.Prepare the Lysogeny Broth (LB) liquid media (1 L) as indicated below: <br/><br />
+ Bacto-Tryptone - 10 g<br/><br />
+ NaCl - 10 g<br/><br />
+ Yeast Extract - 5 g<br/><br />
Add ddH2O and 5mmol/L Tris Buffer in a measuring cylinder to ensure accuracy, to make a total of 1 liter and pH is 8.0.<br/><br />
2.Prepare the Lysogeny Broth (LB) solid media (1 L) as indicated below:<br/><br />
+ Bacto-Tryptone - 10 g<br/><br />
+ NaCl - 10 g<br/><br />
+ Yeast Extract - 5 g<br/><br />
+ Difco Agar - 15g<br/><br />
Add ddH2O and 5mmol/L Tris Buffer in a measuring cylinder to ensure accuracy, to make a total of 1 liter and pH is 8.0.<br/><br />
3. Autoclaving<br/><br />
Autoclave at 121 °C for 60 minutes. After the media cooling down enough, antibiotics Ampicillin(100mg of Ampicillin per 1ml of the media) are added. At last the media are poured 15ml on each plate and become solid.Store the plate at 4℃ refrigerator.<br />
</p><br />
<br />
<font face="Arial, Helvetica"><br />
<p>&nbsp;</p><br />
<br><br />
<a name="Bacterial"></a></font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Bacterial Transformation</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Transformation is commonly used to introduce recombinant plasmid DNA into bacterial strains which can transform naturally or can be made competitive for transformation by artificial means. The purpose of this technique is to introduce a recombinant plasmid DNA into a bacterial strains and to use bacteria strains to amplify the plasmid mutant-pSB1A3 for further plasmid construction.</p><br />
<br />
<strong>Method</strong><br /><br />
1. Take out an appropriate number of tubes that contain competent cells(100μl ) from the freezer. Immediately place the tubes on ice, so that all but the cap is surrounded by ice. Allow the cells to thaw on ice for 2-5 min.<br /><br />
2. Visually check the cells to see whether they have thawed and gently flick the cells 1-2 times to evenly resuspend the cells.<br /><br />
3. Add 10μl PCR products(mini-prep purified) to the competent cells DH-5α. Stir gently to mix and return the tube to the ice, making sure that the tube is surrounded by ice except for the cap. Repeat for additional two times for the same samples.<br /><br />
4. Incubate the tubes on ice for 30 min.<br /><br />
5. Place the tubes in a 42°C water bath for exactly 90 sec; do not shake.<br /><br />
6. Place the tubes on ice for 2 min to cool down.<br /><br />
7. Add 800 l of room temperature LB medium to each tube.<br /><br />
8. Shake the tubes vigorously at 37<a name="OLE_LINK2" id="OLE_LINK2"></a><a name="OLE_LINK1" id="OLE_LINK1">°C</a> for 45-60 min.<br /><br />
9. Centrifuge the tubes at 3K RPM for 1 min. Discard the supernatant liquor and leave 100-200 μl of the mixtures.Mix the contents and spread the whole liquid on LB agar plates containing the appropriate antibiotic ampicillin for the plasmid.<br /><br />
10. Place the plates on the bench for several min to allow excess liquid to be absorbed, and then invert and incubate overnight at 37°C (12-16 h).</p><br />
<br><br />
<a name="Colony"></a></font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Colony PCR for Verification</font> </b></font></h3><br />
<font face="Arial, Helvetica"><br />
<P>Colony PCR is used to identify and select cell colonies that have the correct plasmid inserted. The procedure is a way to do several PCR operations on cell colonies in parallel, to evaluate the results and select the corresponding positive cell colonies. After an overnight growth of E.coli, we can pick up several colonies from the plate and do a colony PCR verification. Besides, the colonies we choose and should also be stored, we can incubate these colonies in one plate after every colony has been marked.</p><br />
<br />
<strong>Method</strong><br /><br />
1. Prepare the sample reaction as indicated below:<br /><br />
Total: 25μl <br /><br />
+ 0.25 μl of Ex Taq polymerase (company:Takara)<br /><br />
+ 2.5 μl of 10× Taq reaction buffer<br /><br />
+ 1.0 μl of R-NPS-F*<br /><br />
+ 1.0 μl of G-SXA-R*<br /><br />
+ 1.0 μl of plasmid mutant-psb1a3<br /><br />
+ 2.0 μl of dNTP( 25mM ) <br /><br />
+ 17.25 μl of ddH2O <br /><br />
Note: The sequences of primers R-NPS-F , G-SXA-R <br /><br />
R-NPS-F 5'-TATAGCGGCCGCCTTAAGTAAGTAAGAGTATACG-3' <br /><br />
G-SXA-R 5'-TCTCCGACTTAAGGGATCCTATAAACGCAG-3'<br /><br />
<br />
<p> 2. Set parameters for PCR to amplify desired products. </p><br />
<br />
<table><br />
<tr><br />
<td>Temperature</td> <td>Time</td> <td>Cycle</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>5min</td> <td>1</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>55˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>72˚C</td> <td>1min 20sec</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>4˚C</td> <td>7hrs</td> <td>1</td><br />
</tr><br />
</table><br />
<br />
3. Electrophorese the total system and observe the lane separation. </P><br />
<br><br />
<a name="Culture"></a></font><br />
<br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Cultivate the Bacteria</b></font></h3><br />
<font face="Arial, Helvetica"><p>According to the results of the PCR detection, positive colonies are chosen and transferred them to 5ml LB liquid media ( 5μl of ampicillin added) stored in 12.5ml centrifuge tubes. Put the centrifuge tubes in 37℃ gas bath overnight. </p><br />
<br><br />
<a name="Plasmid"></a><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Plasmid DNA Isolation</b></font></h3><br />
<p>Use E.Z.N.A.TM Plasmid Mini I to realize plasmid DNA isolation.<br /><br />
<p><strong>Method</strong></p><br />
<br />
<ol><br />
<li>Transfer 5 ml of overnight culture into a 1.5-ml eppendorf tube labeled with group number. </li><br />
<li>Centrifuge the sample at max. speed of desk top centrifuge and RT for 1min to pellet the cells.</li><br />
<li>Discard the supernatant. Remove as much of the supernatant as possible without disturbing the cell pellet. </li><br />
<li>Repeat step 1 and 2 twice. </li><br />
<li>Resuspend the pellet completely in 250 ml of Solution I (containing RNase A) by vortexing the samples vigorously . No clumps should be visible in the tube. </li><br />
<li>Add 250 ml of Solution II and mix the sample by gently inverting the tube 4 to 6 times. Do not vortex or shake the sample vigorously. The bacterial suspension should begin to clear which have lysed the bacterial cells in this step. <strong>Warning: </strong>Do not stop here for more than five min, as the high pH hurts your DNA! </li><br />
<li>Add 350 ml of Solution III and mix by gently inverting the tube 4 to 6 times until a flocculent white precipitate forms. Do not shake vigorously, as it might break the genomic DNA. </li><br />
<li>Centrifuge at maximum speed for 10 min at room temperature to pellet the cell debris. You should see a white precipitate in the tube after the centrifugation. </li><br />
<li>While the samples are centrifuging, for each sample, label a clean HiBind Miniprep Column which is to assembled in a 2-ml collection tube</li><br />
<li>Apply the supernatants from step 8 to the columns. </li><br />
<li>Centrifuge at maximum speed for 1 min at RT. Discard the flow-through in the collection tube. </li><br />
<li>Add 500 ml of Buffer HB to wash the Hibind Miniprep Column. Centrifuge at maximum speed for 1 min at RT. Discard the flow-through in the collection tube. </li><br />
<li>Wash the column by adding 700 ml of DNA Wash Buffer diluted with absolute ethanol. Centrifuge at maximum speed for 1 min at room temperature and discard the flow-through.</li><br />
<li>Then centrifuge the tubes again for 2 min to remove all the moisture.</li><br />
<li>Place the column in a clean 1.5 ml eppendorf tube that is labeled with the plasmid name and group number. To elute the DNA, add 50 ml of Elution Buffer to the center of each column. Let the samples stand for 2 or more minutes at RT, and then centrifuge for 1 min. The sample in the centrifuge tube (bottom) is your plasmid DNA. </li><br />
<li>Discard the column and save the sample in the eppendorf tube by placing it in the freezer (-20°C). </li><br />
</ol><br />
<br />
<a name="#Restriction_2" ></a> </font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Restriction Enzyme Digestion and Electrophoresis</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Because the colony PCR test is so sensitive and affect markedly by environment factors. So we do a restriction enzyme digestion to ensure that the isolated plasmid is the site-directed mutated plasmid.<br /><br />
<strong>Method</strong><br /><br />
1. Prepare the control reaction as indicated below:<br /><br />
Total: 10μl<br /><br />
+ 0.5μl of Pst I restriction enzyme(company :Takara)<br /><br />
+ 1μl of 10XH buffer<br /><br />
+ 1μl of plasmid DNA<br /><br />
+ 7.5μl of ddH2O <br /><br />
2. Prepare the sample reaction as indicated below:<br /><br />
Total: 10μl<br /><br />
+ 0.5μl of Afl II restriction enzyme, (company :Takara)<br /><br />
+ 1μl of 10XM buffer<br /><br />
+ 1μl of plasmid DNA<br /><br />
+ 1.0μl of 0.01% BSA<br /><br />
+ 6.5μl of ddH2O <br /><br />
3. Electrophorese the total system and observe the lane separation.</p><br />
<br><br />
<a name="Amplify"></a> </font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Polymerase Chain Reaction(GFP &amp; RFP) and Electrophoresis</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>GFP and RFP DNA fragments are the insert which need to be ligate to the plasmid mutant-psb1a3. Do a PCR amplification can get enough quantities for the following reactions.<br /><br />
<strong>Method </strong><br /><br />
1.Prepare the sample reaction as indicated below:<br /><br />
Total: 100μl ( PCR <a name="OLE_LINK37" id="OLE_LINK37"></a><a name="OLE_LINK36" id="OLE_LINK36">amplification</a> of GFP fragments) <br /><br />
+ 1.0μl of Taq DNA polymerase,#EP0402<br /><br />
+ 10μl of 10XTaq buffer <br /><br />
+ 10μl of MgCl2(25mM)<br /><br />
+ 10μl of dNTP(2mM)<br /><br />
+ 2μl of G-SXA-R* <br /><br />
+ 2μl of G-SXA-F*<br /><br />
+ 4μl of DNA template<br /><br />
+ 61μl of ddH2O <br /><br />
Total: 100μl(PCR amplification of RFP fragments) <br /><br />
+ 1.0μl of Taq DNA polymerase, EP0402<br /><br />
+ 10μl of 10XTaq buffer <br /><br />
+ 10μl of MgCl2(25mM)<br /><br />
+ 10μl of dNTP(2mM)<br /><br />
+ 2μl of R-NPS-R* <br /><br />
+ 2μl of R-NPS-F*<br /><br />
+ 4μl of DNA template<br /><br />
+ 61μl of ddH2O <br /><br />
<br />
Note: The sequences of primers R-NPS-F , R-NPS-R , G-SXA-F ,G-SXA-R <br /><br />
R-NPS-F 5'-TATAGCGGCCGCCTTAAGTAAGTAAGAGTATACG-3' <br /><br />
R-NPS-R 5'-CGGAGACTAGTCTGCAGATCACATAAGTAAAGTGATAATC-3' <br /><br />
G-SXA-F 5'-CTAGACTAGTTCTAGAGGCGGACTCACTATAGA-3' <br /><br />
G-SXA-R 5'-TCTCCGACTTAAGGGATCCTATAAACGCAG-3'<br /><br />
<br />
<p> 2. Set parameters for PCR to amplify desired products. </p><br />
<br />
<table><br />
<tr><br />
<td>Temperature</td> <td>Time</td> <td>Cycle</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>5min</td> <td>1</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>55˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>72˚C</td> <td>1min 20sec</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>4˚C</td> <td>7hrs</td> <td>1</td><br />
</tr><br />
</table><br />
</p><br />
</font><br />
<ul><br />
<li>Use DNA Gel Extraction Kit to purify the GFP and RFP DNA fragments after the Electrophoresis.</li><br />
</ul><br />
<p align="left"><strong>Figure</strong></p><br />
<p><font face="Arial, Helvetica"><img src="https://static.igem.org/mediawiki/2012/7/72/111.png" alt="" class="img_fl img_border" align="left"/> </font></p><br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<p>(Figure 2 : This figure shows that The PCR reaction system can amplify large quantities of GFP and RFP DNA fragments. The digestion based on the GFP and RFP DNA fragments can be done to prepare for the ligation. Lane 1 represents the template E.coli 817 can amplify the GFP and RFP DNA fragments , lane 2 represents the template E.coli 817(355.5) can also amplify the GFP and RFP DNA fragments.)<br/><br/><br />
<font face="Arial, Helvetica"><br/><br />
<a name="Restriction_Enzyme"></a><br />
</font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Double Restriction Enzyme Digestion and Electrophoresis.</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Use specific restriction enzymes to digest plasmid mutant-psb1a3,GFP and RFP to get sticky ends and purify the DNA fragment after the Electrophoresis.<br /><br />
<strong>Method:</strong></p><br />
</font><br />
<ul><br />
<li> Digestion of plasmid mutant-psb1a3 </li><br />
</ul><br />
<p>Prepare the sample reaction as indicated below:<br /><br />
Total: 50μl <br /><br />
+ 3.0μl of Not I restriction enzyme, #ER0591<br /><br />
+ 3.0μl of Afl II restriction enzyme, #ER0831<br /><br />
+ 5.0μl of 10X buffer O <br /><br />
+ 1.0μl of mutant-psb1a3 plasmid <br /><br />
+ 37.0μl of ddH2O<br /><br />
2. Digestion of PCR product GFP<br /><br />
Prepare the sample reaction as indicated below:<br /><br />
Total: 50μl <br /><br />
+ 3.0μl of Not I restriction enzyme, #ER0591<br /><br />
+ 3.0μl of Spe I restriction enzyme, #ER1251 <br /><br />
+ 5μl of 10X buffer Tango<br /><br />
+ 10μl of PCR products GFP<br /><br />
+ 29μl of ddH2O<br /><br />
3. Digestion of PCR product RFP<br /><br />
Prepare the sample reaction as indicated below:<br /><br />
Total: 50μl <br /><br />
+ 3.0μl of Afl II restriction enzyme, #ER0831<br /><br />
+ 3.0μl of Spe I restriction enzyme, #ER1251 <br /><br />
+ 5μl of 10X buffer Tango<br /><br />
+ 10μl of PCR products RFP<br /><br />
+ 29μl of ddH2O<br /><br />
4. Put the tubes in 37℃ environment for 4-8 hours <br /><br />
5. Use DNA Gel Extraction Kit to purify the mutant-psb1a3 fragment, GFP and RFP after digestion and named them by mutant-psb1a3 (NA) ,GFP(NS) and RFP(AS) after the Electrophoresis.</p><br />
<font face="Arial, Helvetica"><br><br />
<a name="Ligayion"></a> </font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Ligation</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Ligation is the process that target DNA gene is inserted into a plasmid. Both the vector and insert are prepared to have the sticky ends. These two kinds of DNA pieces are placed in a reaction tube and the proper DNA ligase, buffer, and cofactors are added for the reaction to take place. When done properly, the ligation will result in a successful combination of the insert and plasmid into one plasmid.Based on the digestion of mutant-psb1a3,GFP and RFP DNA fragments,also ligation can be done. We ligate mutant-psb1a3 vector and sticky GFP and RFP DNA fragments to construct an new plasmid mutant-psb1a3-GR. <br /><br />
1. Prepare the control reaction as indicated below:<br /><br />
Total: 10μl <br /><br />
+ 1.0μl of plasmid mutant-psb1a3 (NA) <br /><br />
+ 3.0μl of GFP(NS) <br /><br />
+ 3.0μl of RFP(AS) <br /><br />
+ 2.0μl of T4 DNA Ligase,#EL0011 <br /><br />
+ 1.0μl of 10XT4 Ligase buffer<br /><br />
Note: GFP(NS) means the product of GFP DNA fragments digested by restriction enzyme Not I and Spe I. <br /><br />
2. Prepare the sample reaction as indicated below:<br /><br />
Total: 10μl <br /><br />
+ 1.0μl of plasmid mutant-psb1a3 (NA) <br /><br />
+ 2.0μl of T4 DNA Ligase, #EL0011 <br /><br />
+ 1.0μl of 10XT4 Ligase buffer<br /><br />
+ 6.0μl of ddH2O<br /><br />
3. Put the tubes in 22℃ water bath, react for 8-12 hours. <br><br />
<a name="Bacterial_Transformation"></a> </font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Bacterial Transformation</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Transform the ligation products into the DH-5α competent cells, put the plate on 37℃ gas bath for 12-16 hours. </p><br />
<br><br />
<a name="Bacterial_Colony"></a> </font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Bacterial Colony PCR</font></b> </font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Colony PCR is used to identify and select cell colonies that have the correct plasmid insert. This procedure is a way to do several PCR operations on cell colonies in parallel, to evaluate the results and select the corresponding good cell colonies. After an overnight growth of E.coli, we can pick up some colonies from the plate and do a colony PCR verification. Besides, the colonies we choose and should also be stored, we can incubate these colonies in one plate after every colony has been marked.<br /><br />
<strong>Method</strong><br /><br />
1. Prepare the sample reaction as indicated below:<br /><br />
Total: 20μl <br /><br />
+ 0.25 μl of Ex Taq polymerase,#EP0402 <br /><br />
+ 2.0 μl of 10× Taq reaction buffer<br /><br />
+ 1.0 μl of R-NPS-F* <br /><br />
+ 1.0 μl of G-SXA-R*<br /><br />
+ 5.0 μl of bacterial colony<br /><br />
+ 2.0 μl of dNTP( 25mM ) <br /><br />
+ 8.75 μl of ddH2O <br /><br />
Note: The sequences of primers R-NPS-F , R-NPS-R , G-SXA-F ,G-SXA-R <br /><br />
R-NPS-F 5'-TATAGCGGCCGCCTTAAGTAAGTAAGAGTATACG-3' <br /><br />
R-NPS-R 5'-CGGAGACTAGTCTGCAGATCACATAAGTAAAGTGATAATC-3' <br /><br />
G-SXA-F 5'-CTAGACTAGTTCTAGAGGCGGACTCACTATAGA-3' <br /><br />
G-SXA-R 5'-TCTCCGACTTAAGGGATCCTATAAACGCAG-3'<br /><br />
<br />
<p> 2. Set parameters for PCR to amplify desired products. </p> <br />
<table><br />
<tr><br />
<td>Temperature</td> <td>Time</td> <td>Cycle</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>13.5min</td> <td>1</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>55˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>72˚C</td> <td>1min 20sec</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>4˚C</td> <td>7hrs</td> <td>1</td><br />
</tr><br />
</table><br />
<br />
</font><br />
<ul><br />
<li> Electrophorese the total system and observe the lane separation. </li><br />
</ul><br />
<p><strong>Figure</strong></p><br />
<p> <img src="https://static.igem.org/mediawiki/2012/0/07/1111.png" alt="" class="img_fl img_border" align="left"/> </p><br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<p>(Figure 3: The lane on the figure are 2k DNA fragments, it shows the GFP and RFP DNA fragments are ligated to the vectors which were isolated from the bacterial colonies.) </p><br />
<br />
<p><font face="Arial, Helvetica"><br><br />
<a name="Culture_the"></a><br />
</font></p><br />
<font face="Arial, Helvetica"><br />
</font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Cultivate the Bacteria</b></font></h3><br />
<font face="Arial, Helvetica"><p>According to the results of the PCR detection, we choose positive colonies and transfer them to 5ml LB liquid media ( 5μl of ampicillin has added) stored in 12.5ml centrifuge tubes. Put the centrifuge tubes in 37℃ gas bath overnight. </p><br />
<br />
<p><font face="Arial, Helvetica"><a name="Plasmid_DNA"></a> </font></p><br />
<font face="Arial, Helvetica"> </font> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Plasmid DNA Isolation</b></font></h3><br />
<p><font face="Arial, Helvetica">Use E.Z.N.A.TM Plasmid Mini I to isolate the constructed plasmid mutant-psb1a3-GR. </font></p><br />
<br />
<p><font face="Arial, Helvetica"><a name="Restriction_Enzyme" ></a> </font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Restriction Enzyme Digestion and Electrophoresis</b></font></h3><br />
<p>From the last step, we got the certain quantities of isolated plasmids. In this step, we do two restriction enzyme digestion reactions, one to prove that the plasmid is construct correctly ( mutant-psb1a3-GR ), one to get sticky ends preparing for the ligation.<br /><br />
<strong>Method</strong></p><br />
<p>1.Restriction Enzyme Digestion to prove that plasmid is constructed correctly</p><br />
<br />
<p align="left">1.1. Prepare the sample reaction as indicated below:<br /><br />
Total: 10μl<br /><br />
+ 1.0μl of Not I restriction enzyme,#ER0591<br /><br />
+ 1.0μl of Spe I restriction enzyme,#ER1251 <br /><br />
+ 2.0μl of Buffer Tango( 10X )<br /><br />
+ 1.5μl of plasmid mutant-psb1a3-GR<br /><br />
+ 14.5μl of ddH2O <br /><br />
1.2. Electrophorese the total system and observe the lane separation. </p><br />
<br />
<p>2. Restriction Enzyme Digestion to get sticky ends preparing for the ligation.</p><br />
<br />
<p align="left">2.1. Prepare the sample reaction as indicated below:<br /><br />
Total: 50μl<br /><br />
+ 5.0μl of Pst I restriction enzyme, #ER0611<br /><br />
+ 5.0μl of Xba I restriction enzyme, #ER0681 <br /><br />
+ 3.0μl of Buffer Tango( 10X )<br /><br />
+ 5.0μl of plasmid mutant-psb1a3-GR<br /><br />
+ 32.0μl of ddH2O <br /><br />
2.2. Electrophorese the total system and observe the lane separation.<br /><br />
2.3. Cut the gel of specific position and collect it in tubes that have measured weight. <br /><br />
2.4. Use DNA Gel Extraction Ki to purify the plasmid DNA mutant-psb1a3-GR(PX) .<br /><br />
Note: Mutant-psb1a3-GR(PX) means plasmid Mutant-psb1a3-GR digested by Pst I and Xba I.</p><br />
<br />
<p><strong>Figure</strong></p><br />
<p align="left"><strong><img src="https://static.igem.org/mediawiki/2012/d/d8/11111.png" alt="" class="img_fl img_border" align="left" /></strong></p><br />
<p>(Figure 4 : The double digestion of mutant-psb1a3 forms a linear DNA fragments and it runs slower than circle DNA fragments. This suggest that the double digestion of mutant-psb1a3 works in a high efficiency, and desired sticky ends are formed.1,3,5 are plasmids digested by restriction enzyme Pst I and Xba I from different colonies, 2,5,6 are pure plasmid mutant-psb1a3-GR, 7 is the plasmid mutant-psb1a3. )</p><br />
<p align="left">&nbsp;</p><br />
<p><font face="Arial, Helvetica"><a name="Ligation1" ></a> </font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Ligation</b></font></h3><br />
<p>Ligation is the process that target DNA gene is inserted into a plasmid. Both the vector and insert are prepared to have the sticky ends. These two kinds of DNA pieces are placed in a reaction tube and the proper DNA ligase, buffer, and cofactors are added for the reaction to take place. When done properly, the ligation will result in a successful combination of the insert and plasmid into one plasmid.Based on the digestion of mutant-psb1a3-GR, terminator DNA fragments,ligation can be done. We ligate mutant-psb1a3-GR vector and sticky terminator DNA fragments to construct an new plasmid mutant-psb1a3-GR-t.By detecting the quantities of GFP and RFP, terminator efficiency can be calculated. <br /><br />
1. Prepare the control reaction as indicated below:<br /><br />
Total: 10μl <br /><br />
+ 1.0μl of plasmid mutant-psb1a3 (NA) <br /><br />
+ 6.0μl of terminator<br /><br />
+ 2.0μl of T4 DNA Ligase , #EL0011 <br /><br />
+ 1.0μl of 10XT4 Ligase buffer<br /><br />
2. Put the tubes in 22℃ water bath, react for 8-12 hours. </p><br />
<p><font face="Arial, Helvetica"><a name="Bacteria" id="Culture_the"></a> </font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Bacteria transformation</b></font></h3><br />
<p>Transform the ligation products into the DH-5α competent cells, put the plate on 37℃ gas bath for 12-16 hours. </p><br />
<p><font face="Arial, Helvetica"><a name="Cultivate"></a> </font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Cultivate the Bacteria</b></font></h3><br />
<p>According to the results of the PCR detection, we choose positive colonies and transfer them to 5ml LB liquid media ( 5μl of ampicillin has added) stored in 12.5ml centrifuge tubes. Put the centrifuge tubes in 37℃ gas bath overnight. </p><br />
<p><font face="Arial, Helvetica"><a name="Flow" ></a> </font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Flow Cytometer Analysis</b></font></h3><br />
<p> 1. NaCl solution( 0.9% )<br /><br />
2. 75% Methanol<br /><br />
B. Procedures:<br /><br />
1. Transfer overnight suspension culture to 1.5ml centrifuge tubes and centrifuge at 12000RPM for 30s. Pour the supernatant and add overnight suspension culture and centrifuge again until enough bacteria has been collected. <br /><br />
2. Use NaCl solution(0.9%) to mix the bacteria and vibrate the centrifuge tubes until the bacteria distributed uniformly.<br /><br />
3. Put the centrifuge tubes into the Flow Cytometer and set parameters and run the program.</p><br />
<p>&nbsp;</p><br />
<p><font face="Arial, Helvetica"><a name="Fluorescence"></a> </font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Fluorescence Microscope</b></font></h3><br />
<p><strong>Method</strong><br /><br />
Add 10μl of former bacteria solution to micro slide and cover with coverslip.Then placed it on the Fluorescence Microscope with 488nm light activating and observe the GFP and RFP. </p><br />
<br> <br />
<br />
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</html></div>M.B.ZHOUhttp://2012.igem.org/Team:SUSTC-Shenzhen-B/protocol1Team:SUSTC-Shenzhen-B/protocol12012-10-26T20:32:27Z<p>M.B.ZHOU: </p>
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#catlinks {display:none; }<br />
#footer-box {display:none; }<br />
#menubar {display:none; }<br />
<br />
/* googleapis-font */<br />
@font-face {<br />
font-family: 'Open Sans';<br />
font-style: normal;<br />
font-weight: 700;<br />
src: local('Open Sans Bold'), local('OpenSans-Bold'), url(http://themes.googleusercontent.com/static/fonts/opensans/v6/k3k702ZOKiLJc3WVjuplzHhCUOGz7vYGh680lGh-uXM.woff) format('woff');<br />
}<br />
@font-face {<br />
font-family: 'Open Sans';<br />
font-style: normal;<br />
font-weight: 600;<br />
src: local('Open Sans Semibold'), local('OpenSans-Semibold'), url(http://themes.googleusercontent.com/static/fonts/opensans/v6/MTP_ySUJH_bn48VBG8sNSnhCUOGz7vYGh680lGh-uXM.woff) format('woff');<br />
}<br />
@font-face {<br />
font-family: 'Open Sans';<br />
font-style: normal;<br />
font-weight: 400;<br />
src: local('Open Sans'), local('OpenSans'), url(http://themes.googleusercontent.com/static/fonts/opensans/v6/cJZKeOuBrn4kERxqtaUH3T8E0i7KZn-EPnyo3HZu7kw.woff) format('woff');<br />
}<br />
<br />
/* inner */<br />
.pagenavi{clear:both;padding:0}.pagenavi a,.pagenavi a:visited{background:#dfddc7;display:inline-block;margin:0 8px 0 0;padding:3px 10px;color:#727272}.pagenavi a:hover{background:#dfddc7;display:inline-block;margin:0 8px 0 0;padding:3px 10px;color:#b03121;text-decoration:none}.pagenavi .current{background:#dfddc7;display:inline-block;margin:0 8px 0 0;padding:3px 10px;color:#b03121}.pagenavi .pages{background:#dfddc7;display:inline-block;margin:0 8px 0 0;padding:3px 10px}#breadcrumb{text-align:right;margin-bottom:34px;clear:right}.post{margin-bottom:30px;padding-bottom:30px;clear:both;border-bottom:1px solid #dfddc7}.post img{padding:5px;background:#d2d0ba;width:590px;height:236px}.single{padding-bottom:20px}.postimg{margin-bottom:20px}.posttitle{margin:0 0 5px 0}.posttitle,.posttitle a{font-size:18px;color:#3f4432}.posttitle a:hover{text-decoration:none}.entry-text{overflow:hidden;padding-left:30px}.entry-text h2{padding-top:5px}.entry-content{margin:0;padding:12px 0 5px 0}.entry-date{float:left;text-align:center;font-size:18px;width:56px;height:48px;padding-top:8px;line-height:normal;-moz-border-radius:56px;-webkit-border-radius:56px;-khtml-border-radius:56px;border-radius:56px;background:#3f4432;color:#f1efda;display:block}.month{font-size:11px;display:block}.entry-utility{padding:20px 0 0;font-size:11px;font-style:italic}.single .entry-utility{padding:0 0 0}#comment h2{margin-bottom:25px;text-transform:uppercase;font-size:14px}.commentlist{list-style-type:none;padding:0;margin:0}.commentlist ol{list-style-type:none;padding:30px 0 0 90px;margin:0}.commentlist li{position:relative;padding:0 0 30px 0}.commentlist li li{position:relative;padding:0}.avatar{position:absolute;top:0;left:0}.fn{font-size:12px;font-style:normal}.tdate{padding-left:30px}.tdate,.reply{font-size:11px;color:#a6a6a6;font-style:italic}.comment-body{margin:0 0 0 90px;padding:20px;background:#f7f5e3}.comment-body p{margin-bottom:5px;margin-top:10px}.comment-body .more{padding:0 0}#commentform{margin-bottom:15px}#commentform label{display:block}#commentform .text-input{margin-bottom:8px;padding:8px 5px;vertical-align:middle}#commentform .textarea{margin-bottom:20px;padding:8px 5px;vertical-align:top;width:90%}.ts-gallery-img img{max-width:100%;padding:0;margin:0 auto}.ts-gallery-clear{clear:both;height:1px!important;line-height:1px!important;float:none!important}.ts-gallery ul{list-style-type:none;margin:0;padding:0;clear:both}.ts-gallery ul li.nomargin{margin-right:0}.ts-gallery-text{padding:3px 0;text-align:center}.ts-gallery-text h2{font-size:13px;color:#3f4432;margin-bottom:20px;font-weight:600;font-family:'Open Sans',sans-serif}.no-gallery-text{display:none}.ts-gallery-img{background:#d2d0ba;padding:5px;position:relative;line-height:normal}.ts-gallery-img:hover{background:#3f4432}.ts-gallery-img a.image{display:block;position:relative;overflow:hidden;line-height:normal}.ts-gallery-img a .rollover{background:url(/wiki/images/5/5d/Sustc-b1-hover-zoom.png);background-color:#000;background-repeat:no-repeat;background-position:center;display:block;position:absolute;z-index:10;display:none;cursor:pointer}.ts-gallery-img a .rollover.gotopost{background:url(/wiki/images/e/ec/Sustc-b1-hover-doc.png);background-color:#000;background-repeat:no-repeat;background-position:center}.ts-gallery-col4{list-style-type:none;padding:0;margin:0}.ts-gallery-col4 li{list-style-type:none;padding:0;margin:0 20px 0 0;width:220px;float:left}.ts-gallery-col4 li.nomargin{margin-right:0}.ts-gallery-col4 .ts-gallery-img{width:210px;height:81px}.ts-gallery-col4 .ts-gallery-img img{width:210px;height:81px}.ts-gallery-col4 .ts-gallery-img a.image{width:210px;height:81px;display:block;position:relative}.ts-gallery-col4 .ts-gallery-img a .rollover{width:210px;height:81px}.ts-gallery-col3{list-style-type:none;padding:0;margin:0}.ts-gallery-col3 li{list-style-type:none;padding:0;margin:0 20px 25px 0;width:300px;float:left}.ts-gallery-col3 li.nomargin{margin-right:0}.ts-gallery-col3 h2{margin:0 0 5px 0!important}.ts-gallery-col3 .ts-gallery-img{width:290px;height:115px}.ts-gallery-col3 .ts-gallery-img img{width:290px;height:115px}.ts-gallery-col3 .ts-gallery-img a.image{width:290px;height:115px;display:block;position:relative}.ts-gallery-col3 .ts-gallery-img a .rollover{width:290px;height:115px}.ts-gallery-col3 .ts-gallery-text{padding:5px 0 0 0}form{margin:0;padding:0}fieldset{border:0}#contactform{margin:0 auto;position:relative}#contactform h4{text-transform:uppercase;margin-bottom:20px}#contactform label{display:block;width:100%;float:left;padding-bottom:5px}span.required{color:#888}span.error{color:red;text-align:left;font-size:11px;padding-bottom:15px;display:block}#contactform input.text-input{margin-bottom:15px;vertical-align:middle;width:60%;float:left;font-style:italic;padding:8px;color:#727272;font-size:11px}#contactform textarea{width:95%;float:left;font-style:italic;color:#727272;font-size:11px;font-family:Arial,Helvetica,sans-serif}#message{margin-left:0;font-weight:700;color:red}#message h2{}#message p{margin:6px 0}.note{color:#d45454}#contactform .button{cursor:pointer;margin-top:20px;clear:both;float:left}<br />
<br />
/* style */<br />
html,body{height:100%}body{font-family:Arial,Helvetica,sans-serif;font-size:12px;color:#727272;margin:0;padding:0;line-height:20px;background:#d6d4c0 url(/wiki/images/d/d7/Sustc-b1-pattern.png) repeat}*{margin:0;padding:0}:focus{outline:0}form{margin:0;padding:0}hr{border-width:0;height:1px;line-height:0;margin:30px 0;page-break-after:always;text-align:center;width:100%;clear:both;color:#dfddc7;background-color:#dfddc7}.clearfix:before,.clearfix:after{content:'\0020';display:block;overflow:hidden;visibility:hidden;width:0;height:0}.clearfix:after{clear:both}.clearfix{zoom:1}.clear,.clr{clear:both;display:block;overflow:hidden;visibility:hidden;width:0;height:0}h1,h2{margin-bottom:25px}h3,h4,h5,h6{margin-bottom:10px}h1{font-size:26px}h2{font-size:20px}h3{font-size:16px}h4{font-size:14px}h5{font-size:12px}h6{font-size:10px}h1,h2{font-weight:400;font-family:'Open Sans',sans-serif;color:#404631}h3,h4,h5,h6{font-weight:600;font-family:'Open Sans',sans-serif;color:#404631}.pagetitle{margin-bottom:34px;float:left}a,a:visited,.colortext{text-decoration:none;font-weight:400;color:#af3728}a:hover{text-decoration:underline;color:#af3728}a img{border:0}.alignleft,img.alignleft{display:inline;float:left;margin-right:20px}.alignright,img.alignright{display:inline;float:right;margin-left:20px}.aligncenter,img.aligncenter{clear:both;display:block;margin-left:auto;margin-right:auto}.alignnone,img.alignnone{clear:both;display:block;margin-left:auto;margin-right:auto}img.alignleft,img.alignright,img.aligncenter,img.alignnone{margin-bottom:12px}img{max-width:100%;height:auto}.frame{padding:5px;background:#d2d0ba}.row h4{color:#b03121;padding:15px 0}p,ul,ol,blockquote{margin-bottom:20px}ul{list-style:square;margin:0 0 18px 1.5em}ol{list-style:decimal;margin:0 0 18px 2.2em}ol ol{list-style:upper-alpha}ol ol ol{list-style:lower-roman}ol ol ol ol{list-style:lower-alpha}ul ul,ol ol,ul ol,ol ul{margin-bottom:0}blockquote{margin:0 0 20px 0;padding:0 10px 0 50px;background-image:url(/wiki/images/3/3b/Sustc-b1-quote.png);background-repeat:no-repeat;background-position:0 0;clear:both;font-family:Georgia,Arial;font-style:italic;font-size:16px;line-height:22px}blockquote.left,blockquote.right{float:right;letter-spacing:0;margin-bottom:20px;margin-left:20px;margin-top:0;padding:0 20px 10px 60px;width:43%;background-position:0 0}blockquote.left{float:left;margin-left:0;margin-right:20px}blockquote p{margin-bottom:0;font-size:16px;line-height:20px}code{font-family:Verdana,Arial;letter-spacing:1px;margin:25px 0 25px 0;display:block;font-size:.9em;border-left:4px solid #cfcfcf;padding:15px 10px}#bodychild{width:1000px;margin:0 auto;padding:50px 0}#outercontainer{width:1000px}#outerheader{-webkit-border-top-left-radius:8px;-webkit-border-top-right-radius:8px;-moz-border-radius-topleft:8px;-moz-border-radius-topright:8px;border-top-left-radius:8px;border-top-right-radius:8px}#outerfooter{border-top:1px solid #e0e0e0;-webkit-border-bottom-left-radius:8px;-webkit-border-bottom-right-radius:8px;-moz-border-radius-bottomleft:8px;-moz-border-radius-bottomright:8px;border-bottom-left-radius:8px;border-bottom-right-radius:8px}#outerheader,#outerslider,#outerbeforecontent,#outermain{width:100%;margin:0 auto;background:#f1efda}#slidercontainer,#beforecontent,#maincontent,#footer{width:940px;margin:0 auto}header{padding:0}#logo.frontpage,#logo.frontpage:before{border:0!important}#logo{border-bottom:1px solid #dfddc7;margin-bottom:25px;padding-bottom:30px;position:relative;z-index:10}#logo:before{border-bottom:1px solid #dfddc7;bottom:2px;content:"";display:block;left:0;position:absolute;right:0;top:0;z-index:-1}#logo{margin:0 30px;padding:30px 0;text-align:center}#logo img{display:inline-block}#sn{list-style-type:none;margin:0;padding:25px 0 0 0;float:right}#sn li{list-style-type:none;margin:0;padding:0 10px 0 20px;display:inline;background:transparent}#sn span{height:20px;width:20px;display:inline;display:inline-block}.icon-img{background-position:0 0}.icon-img:hover{background-position:0 -20px!important}#navigation{float:none;clear:both;padding:0 30px;height:70px;-webkit-border-top-left-radius:7px;-webkit-border-top-right-radius:7px;-moz-border-radius-topleft:7px;-moz-border-radius-topright:7px;border-top-left-radius:7px;border-top-right-radius:7px;background:#494f3a;background:-webkit-gradient(linear,left top,left bottom,from( #4e543d),to( #404533));background:-moz-linear-gradient(top, #4e543d, #404533)}.nav-shadow{background:url(/wiki/images/c/cb/Sustc-b1-nav-shadow.png) repeat bottom;height:6px}nav{position:relative;z-index:9000;float:none;margin:0}#topnav{margin:0;padding:0;list-style-type:none;overflow:visible;position:relative;float:left;font-size:12px}.sf-menu a{text-decoration:none;display:block;position:relative;padding:14px 25px;text-decoration:none;font-weight:400;text-transform:uppercase;color:#f1efda;font-weight:400;font-family:'Open Sans',sans-serif}.sf-menu>li:first-child a{padding-left:0}.sf-menu>li{padding-left:2px;position:relative;z-index:10;border-right:1px solid #575f46}.sf-menu>li:before{bottom:0;content:"";display:block;left:0;position:absolute;right:0;top:0;z-index:-1;border-right:1px solid #3c412f}.sf-menu a:hover,.sf-menu li a.current{color:#a8a581}.sf-menu li a{line-height:42px}.sf-menu ul a:hover{}.sf-menu li li{text-align:left;line-height:20px;margin:0}.sf-menu,.sf-menu *{margin:0;padding:0;list-style:none}.sf-menu{line-height:100%;position:absolute;right:0;bottom:0;float:left}.sf-menu ul{position:absolute;top:-999em;width:14em}.sf-menu ul li{width:100%}.sf-menu li:hover{visibility:inherit}.sf-menu li{float:left;position:relative;margin:0}.sf-menu li li{margin:0 0}.sf-menu li:hover ul,.sf-menu li.sfHover ul{left:0;top:6em;z-index:99}ul.sf-menu li:hover li ul,ul.sf-menu li.sfHover li ul{top:-999em}ul.sf-menu li li:hover ul,ul.sf-menu li li.sfHover ul{left:14em;top:-1px;margin-left:0}ul.sf-menu li li:hover li ul,ul.sf-menu li li.sfHover li ul{top:-999em}ul.sf-menu li li li:hover ul,ul.sf-menu li li li.sfHover ul{left:14em;top:-1px}.sf-menu ul li a{padding:10px 25px!important;text-transform:none;line-height:normal;font-size:13px!important;display:block;width:auto;white-space:no-wrap}.sf-menu ul li a:hover{}.sf-menu li ul{padding:0;-ms-filter:"alpha(Opacity=50)";filter:alpha(opacity=80);-moz-opacity:.8;-khtml-opacity:.8;opacity:.8}.sf-menu a.sf-with-ul{min-width:1px}.sf-sub-indicator{position:absolute;display:block;right:10px;top:1.05em;width:10px;height:10px;text-indent:-999em;overflow:hidden}.sf-menu li li{background:#404533;border-bottom:dotted 1px #707563;border-left:5px solid #404533}.sf-menu li li:hover{border-left:5px solid #a8a581;color:#a8a581}#outerslider{padding-bottom:11px}#slidercontainer{position:relative;padding:0;background:#e1dfc9}#slider{position:relative}.jcarousel-container{overflow:hidden;width:785px;height:107px;margin:20px auto 0 auto;position:relative;clear:both;padding:0}.jcarousel-clip{z-index:2;padding:0;margin:0;overflow:hidden;position:relative}.jcarousel-list{z-index:1;overflow:hidden;position:relative;top:0;left:0;margin:0;padding:0}.jcarousel-item{float:left;list-style:none;width:185px;margin-right:15px}#feature_gallery{width:940px;padding:0;margin:0;overflow:hidden}ul#feature_gallery_pager{display:block;overflow:hidden;list-style-type:none;margin:0;padding:0}#feature_gallery ul.menu li a:hover{}ul#feature_gallery_pager li a{overflow:hidden;float:left;width:175px;height:66px;padding:5px;background:#d2d0ba;display:block}ul#feature_gallery_pager li{}#feature_gallery ul.menu a.activeSlide{background:#3f4432}#feature_gallery .bigimgs{width:940px;height:388px;margin:0}#feature_gallery .bigimg{width:940px;height:388px;display:none}#feature_gallery img.change{width:940px}#feature_gallery img.thumb{width:175px;height:66px}.slidedesc{background:url(/wiki/images/4/4b/Sustc-b1-bg-opacityblack.png);padding:10px;width:920px;z-index:100;bottom:0;position:absolute;margin:0;color:#fff}#pager-container{text-align:right;font-size:11px;position:relative}#pager-container a,#pager-container a:visited{padding:0;cursor:pointer;float:left;width:12px;height:22px;display:block;text-indent:-9999px}#pager-container a:hover{text-decoration:none}#pager-container a#mycarousel-prev:hover{background:url(/wiki/images/8/8b/Sustc-b1-button-prevnext.png) no-repeat 0 -22px}#pager-container a#mycarousel-next:hover{background:url(/wiki/images/8/8b/Sustc-b1-button-prevnext.png) no-repeat -12px -22px}#pager-container a#mycarousel-prev{background:url(/wiki/images/8/8b/Sustc-b1-button-prevnext.png) no-repeat 0 0;position:absolute;left:46px;bottom:58px}#pager-container a#mycarousel-next{background:url(/wiki/images/8/8b/Sustc-b1-button-prevnext.png) no-repeat -12px 0;right:46px;bottom:58px;position:absolute}#outerbeforecontent{clear:both}#beforecontent{}#beforethecontent{}.box{float:left;margin-right:2px;width:33.16%;background:#e1dfc9;text-align:center;padding-bottom:28px}.box h2{color:#efeed9;font-weight:700;background:#3f4432;padding:15px 0}.box p{overflow:hidden;padding:0 30px}#outermain{padding:0}#maincontent{}#mainthecontent{padding:32px 0 40px 0}#t-content{width:600px;float:left}#t-content.positionright{float:right}#t-content.positionleft{float:left}.small{font-size:11px;font-style:italic;margin-bottom:5px;display:block;margin-top:-5px}form{margin:0;padding:0}input[type="text"],textarea,input[type="password"],select{font-size:12px;padding:7px;background:#ebe9d1;border:0;color:#727272;border:0}textarea{width:90%}select{font-size:11px;padding:4px 5px}.button,.button:visited,input[type="submit"]{background:#b03121;color:#efeed9;font-size:12px;display:inline-block;padding:6px 13px;cursor:pointer;font-family:Arial,Helvetica,sans-serif;border:0}.button:hover,input[type="submit"]:hover{background:#3f4432;color:#efeed9;cursor:pointer;text-decoration:none}.separator{display:block;height:30px;padding:10px 0;text-align:center;width:100%;clear:both}.separator.line{display:block;text-align:center;width:100%;clear:both;padding:0;border-top:1px solid #dfddc7;margin:30px 0 40px 0;height:1px}.one_half,.one_third,.two_third,.three_fourth,.one_fourth,.one_fifth,.two_fifth,.three_fifth,.four_fifth,.one_sixth,.five_sixth{margin-right:4%;margin-left:0;position:relative;float:left}.one_half{width:48%}.one_third{width:30.6666%}.one_fourth{width:22%}.one_fifth{width:16.8%}.one_sixth{width:13.3333%}.two_third{width:65.3332%}.two_fourth{width:48%}.two_fifth{width:37.6%}.two_sixth{width:30.6666%}.three_fourth{width:74%}.three_fifth{width:58.4%}.three_sixth{width:47.9998%}.four_fifth{width:79.2%}.four_sixth{width:65.3332%}.five_sixth{width:82.6665%}.firstcols{margin-left:0!important}.last,.lastcols{margin-right:0!important;clear:right}.one.column{width:60px}.two.columns{width:140px}.three.columns{width:220px}.four.columns{width:300px}.five.columns{width:380px}.six.columns{width:460px}.seven.columns{width:540px}.eight.columns{width:620px}.nine.columns{width:700px}.ten.columns{width:780px}.eleven.columns{width:860px}.twelve.columns{width:940px}.column,.columns{float:left;display:inline;margin-left:10px;margin-right:10px}table{border-collapse:separate;border-spacing:0;width:100%;margin-bottom:18px}table,td,th{text-align:center}th{padding:10px;text-transform:uppercase;border-bottom:1px solid #dfddc7}td{padding:10px}tfoot td{border:0}th,tr:hover{}table{border:1px solid #dfddc7;border-bottom:0;text-align:left;margin:0 -1px 24px 0;width:100%}tr th,thead th{font-size:12px;font-weight:700;line-height:18px;padding:9px 24px;background:#3f4432;color:#efeed9}tr td{border-bottom:1px solid #dfddc7;padding:6px 24px}tr.odd td{background:#F2F7FC}.pullquote-right,.pullquote-left{padding:0 10px 0 50px;background-image:url(/wiki/images/3/3b/Sustc-b1-quote.png);background-repeat:no-repeat;background-position:0 0;float:right;font-style:italic;font-size:16px;letter-spacing:0;line-height:22px;margin:0 2px 20px 20px;width:50%}.pullquote-left{float:left;margin-left:2px;margin-right:20px}.pullquote{font-family:Georgia,"Times New Roman",Times,serif;font-size:18px;font-style:italic;line-height:28px}.dropcap1{text-shadow:1px 1px 0 #ededed;display:block;float:left;font-size:35px;line-height:35px;margin:2px 8px 0 0;color:#404631}.dropcap2{display:block;float:left;font-size:35px;line-height:45px;width:47px;-moz-border-radius:47px;-webkit-border-radius:47px;-khtml-border-radius:47px;border-radius:47px;float:left;text-align:center;margin:8px 15px 0 0;padding-top:0;background:#3f4432;color:#f1efda}.dropcap3{background:#3f4432;color:#f1efda;border:solid 1px #efefef;display:block;float:left;font-size:35px;line-height:40px;width:47px;height:40px;text-align:center;margin:6px 8px 0 0;padding:5px 0}.dropcap4{display:block;float:left;font-size:15px;line-height:36px;width:36px;-moz-border-radius:36px;-webkit-border-radius:36px;-khtml-border-radius:36px;border-radius:36px;float:left;text-align:center;margin:8px 15px 0 0;padding-top:0;background:#3f4432;color:#f1efda}.highlight1{padding:2px 5px;background-color:#404631;border:solid 1px #ebebeb;color:#fff}.highlight2{padding:2px 5px;background-color:#e1dfc9;border:solid 1px #e1dfc9}.bullet{list-style-type:none;margin:0;padding:0}.bullet 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Sans',sans-serif;-webkit-border-top-left-radius:3px;-webkit-border-top-right-radius:3px;-moz-border-radius-topleft:3px;-moz-border-radius-topright:3px;border-top-left-radius:3px;border-top-right-radius:3px}.tab-content{padding:20px 0}ul.tabs li:hover{}ul.tabs li.active{}html ul.tabs li.active a{color:#b03121;background:#e1dfc9;-webkit-border-top-left-radius:3px;-webkit-border-top-right-radius:3px;-moz-border-radius-topleft:3px;-moz-border-radius-topright:3px;border-top-left-radius:3px;border-top-right-radius:3px}#tab-body{padding:0 20px;background:#e1dfc9;border:0;-webkit-border-radius:3px;-webkit-border-top-left-radius:0;-moz-border-radius:3px;-moz-border-radius-topleft:0;border-radius:3px;border-top-left-radius:0}#toggle{margin:0 0 20px 0}h2.trigger{padding:0 0;margin:0;font-size:13px;background:transparent;font-family:arial;color:#3f4432}h2.trigger span{text-decoration:none;display:block;background:url(/wiki/images/9/93/Sustc-b1-toggle.png) no-repeat 0 5px;padding-left:35px;cursor:pointer;line-height:35px}h2.active span{background:url(/wiki/images/c/cd/Sustc-b1-toggle-down.png) no-repeat 0 5px}h2.trigger a:hover{color:#efeed9}h2.active{background:transparent;color:#af3728}.toggle_container{margin:0 0 1px 0;padding:5px 15px;overflow:hidden;clear:both;background:transparent}.toggle_container .block{padding:0 0 0 20px}.toggle_container .block p{padding-bottom:10px;margin:0}#sidebar{width:300px;float:left;padding:0 0 0 40px}#sidebar.positionleft{float:left;padding:0 40px 0 0}#sidebar.positionright{float:right}#sidebar .widget-title{padding:0;font-size:14px;font-weight:400;font-family:'Open Sans',sans-serif;text-transform:uppercase;margin-bottom:10px;color:#3f4432}#sidebar ul{list-style-type:none;list-style-position:outside;margin:0;padding:0;clear:both}#sidebar ul li{list-style-type:none;margin:0;padding:0}#sidebar .widget-container{margin-bottom:28px;padding-top:28px;background:url(/wiki/images/7/79/Sustc-b1-line.gif) no-repeat}#sidebar .widget-container:first-child{background:0;padding:0}#sidebar li li{list-style-type:none;margin:0 0 3px 0;padding:0 0 3px 0}#sidebar li li a{color:#777}#sidebar li li a:hover{text-decoration:none;color:#af3728}#sidebar ul.sub-menu,#sidebar ul.children,#sidebar ul ul ul{margin:5px 0 0 10px}#sidebar ul.sub-menu li,#sidebar ul.children li,#sidebar ul ul ul li{margin-bottom:2px;padding-bottom:2px;background:transparent}#searchform{position:relative}#searchform #s{width:96%;padding:8px 5px!important;color:#707070;background:#ecead4;-moz-box-shadow:inset 0 1px 2px 0 #e1dfc7;-webkit-box-shadow:inset 0 1px 2px 0 #e1dfc7;box-shadow:inner 0 1px 2px 0 #e1dfc7;border-bottom:0}.rp-widget li{clear:left;margin-bottom:0;padding-bottom:10px}.rp-widget img{padding:4px}.rp-widget li h3{margin-bottom:0}.rp-widget li h3 a{font-size:13px;font-weight:600}.rp-widget li .smalldate{display:block;font-size:11px;font-style:italic;overflow:hidden}#footercontainer{background:#3f4432;-webkit-border-bottom-left-radius:7px;-webkit-border-bottom-right-radius:7px;-moz-border-radius-bottomleft:7px;-moz-border-radius-bottomright:7px;border-bottom-left-radius:7px;border-bottom-right-radius:7px}#footer{padding:25px 0 25px 0;color:#bbb9a1;font-weight:400;font-family:'Open Sans',sans-serif;font-size:13px}#footer a,#footer a:visited{color:#bbb9a1}<br />
<br />
/* prettyPhoto.css */<br />
div.pp_default .pp_top,div.pp_default .pp_top .pp_middle,div.pp_default .pp_top .pp_left,div.pp_default .pp_top .pp_right,div.pp_default .pp_bottom,div.pp_default .pp_bottom .pp_left,div.pp_default .pp_bottom .pp_middle,div.pp_default .pp_bottom .pp_right{height:13px}div.pp_default .pp_top .pp_left{background:url(../images/default/sprite.png) -78px -93px no-repeat}div.pp_default .pp_top .pp_middle{background:url(../images/default/sprite_x.png) top left repeat-x}div.pp_default .pp_top .pp_right{background:url(../images/default/sprite.png) -112px -93px no-repeat}div.pp_default .pp_content .ppt{color:#f8f8f8}div.pp_default .pp_content_container .pp_left{background:url(../images/default/sprite_y.png) -7px 0 repeat-y;padding-left:13px}div.pp_default .pp_content_container .pp_right{background:url(../images/default/sprite_y.png) top right repeat-y;padding-right:13px}div.pp_default .pp_next:hover{background:url(../images/default/sprite_next.png) center right no-repeat;cursor:pointer}div.pp_default .pp_previous:hover{background:url(../images/default/sprite_prev.png) center left no-repeat;cursor:pointer}div.pp_default .pp_expand{background:url(../images/default/sprite.png) 0 -29px no-repeat;cursor:pointer;height:28px;width:28px}div.pp_default .pp_expand:hover{background:url(../images/default/sprite.png) 0 -56px no-repeat;cursor:pointer}div.pp_default .pp_contract{background:url(../images/default/sprite.png) 0 -84px no-repeat;cursor:pointer;height:28px;width:28px}div.pp_default .pp_contract:hover{background:url(../images/default/sprite.png) 0 -113px no-repeat;cursor:pointer}div.pp_default .pp_close{background:url(../images/default/sprite.png) 2px 1px no-repeat;cursor:pointer;height:30px;width:30px}div.pp_default .pp_gallery ul li a{background:url(../images/default/default_thumb.png) center center #f8f8f8;border:1px solid #aaa}div.pp_default .pp_social{margin-top:7px}div.pp_default .pp_gallery a.pp_arrow_previous,div.pp_default .pp_gallery a.pp_arrow_next{left:auto;position:static}div.pp_default .pp_nav .pp_play,div.pp_default .pp_nav .pp_pause{background:url(../images/default/sprite.png) -51px 1px no-repeat;height:30px;width:30px}div.pp_default .pp_nav .pp_pause{background-position:-51px -29px}div.pp_default a.pp_arrow_previous,div.pp_default a.pp_arrow_next{background:url(../images/default/sprite.png) -31px -3px no-repeat;height:20px;margin:4px 0 0;width:20px}div.pp_default a.pp_arrow_next{background-position:-82px -3px;left:52px}div.pp_default .pp_content_container .pp_details{margin-top:5px}div.pp_default .pp_nav{clear:none;height:30px;position:relative;width:110px}div.pp_default .pp_nav .currentTextHolder{color:#999;font-family:Georgia;font-size:11px;font-style:italic;left:75px;line-height:25px;margin:0;padding:0 0 0 10px;position:absolute;top:2px}div.pp_default .pp_close:hover,div.pp_default .pp_nav .pp_play:hover,div.pp_default .pp_nav .pp_pause:hover,div.pp_default .pp_arrow_next:hover,div.pp_default .pp_arrow_previous:hover{opacity:.7}div.pp_default .pp_description{font-size:11px;font-weight:700;line-height:14px;margin:5px 50px 5px 0}div.pp_default .pp_bottom .pp_left{background:url(../images/default/sprite.png) -78px -127px no-repeat}div.pp_default .pp_bottom .pp_middle{background:url(../images/default/sprite_x.png) bottom left repeat-x}div.pp_default .pp_bottom .pp_right{background:url(../images/default/sprite.png) -112px -127px no-repeat}div.pp_default .pp_loaderIcon{background:url(../images/default/loader.gif) center center no-repeat}div.light_rounded .pp_top .pp_left{background:url(../images/light_rounded/sprite.png) -88px -53px no-repeat}div.light_rounded .pp_top .pp_right{background:url(../images/light_rounded/sprite.png) -110px -53px no-repeat}div.light_rounded .pp_next:hover{background:url(../images/light_rounded/btnNext.png) center right no-repeat;cursor:pointer}div.light_rounded .pp_previous:hover{background:url(../images/light_rounded/btnPrevious.png) center left no-repeat;cursor:pointer}div.light_rounded .pp_expand{background:url(../images/light_rounded/sprite.png) -31px -26px no-repeat;cursor:pointer}div.light_rounded .pp_expand:hover{background:url(../images/light_rounded/sprite.png) -31px -47px no-repeat;cursor:pointer}div.light_rounded .pp_contract{background:url(../images/light_rounded/sprite.png) 0 -26px no-repeat;cursor:pointer}div.light_rounded .pp_contract:hover{background:url(../images/light_rounded/sprite.png) 0 -47px no-repeat;cursor:pointer}div.light_rounded .pp_close{background:url(../images/light_rounded/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:75px}div.light_rounded .pp_nav .pp_play{background:url(../images/light_rounded/sprite.png) -1px -100px no-repeat;height:15px;width:14px}div.light_rounded .pp_nav .pp_pause{background:url(../images/light_rounded/sprite.png) -24px -100px no-repeat;height:15px;width:14px}div.light_rounded .pp_arrow_previous{background:url(../images/light_rounded/sprite.png) 0 -71px no-repeat}div.light_rounded .pp_arrow_next{background:url(../images/light_rounded/sprite.png) -22px -71px no-repeat}div.light_rounded .pp_bottom .pp_left{background:url(../images/light_rounded/sprite.png) -88px -80px no-repeat}div.light_rounded .pp_bottom .pp_right{background:url(../images/light_rounded/sprite.png) -110px -80px no-repeat}div.dark_rounded .pp_top .pp_left{background:url(../images/dark_rounded/sprite.png) -88px -53px no-repeat}div.dark_rounded .pp_top .pp_right{background:url(../images/dark_rounded/sprite.png) -110px -53px no-repeat}div.dark_rounded .pp_content_container .pp_left{background:url(../images/dark_rounded/contentPattern.png) top left repeat-y}div.dark_rounded .pp_content_container .pp_right{background:url(../images/dark_rounded/contentPattern.png) top right repeat-y}div.dark_rounded .pp_next:hover{background:url(../images/dark_rounded/btnNext.png) center right no-repeat;cursor:pointer}div.dark_rounded .pp_previous:hover{background:url(../images/dark_rounded/btnPrevious.png) center left no-repeat;cursor:pointer}div.dark_rounded .pp_expand{background:url(../images/dark_rounded/sprite.png) -31px -26px no-repeat;cursor:pointer}div.dark_rounded .pp_expand:hover{background:url(../images/dark_rounded/sprite.png) -31px -47px no-repeat;cursor:pointer}div.dark_rounded .pp_contract{background:url(../images/dark_rounded/sprite.png) 0 -26px no-repeat;cursor:pointer}div.dark_rounded .pp_contract:hover{background:url(../images/dark_rounded/sprite.png) 0 -47px no-repeat;cursor:pointer}div.dark_rounded .pp_close{background:url(../images/dark_rounded/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:75px}div.dark_rounded .pp_description{color:#fff;margin-right:85px}div.dark_rounded .pp_nav .pp_play{background:url(../images/dark_rounded/sprite.png) -1px -100px no-repeat;height:15px;width:14px}div.dark_rounded .pp_nav .pp_pause{background:url(../images/dark_rounded/sprite.png) -24px -100px no-repeat;height:15px;width:14px}div.dark_rounded .pp_arrow_previous{background:url(../images/dark_rounded/sprite.png) 0 -71px no-repeat}div.dark_rounded .pp_arrow_next{background:url(../images/dark_rounded/sprite.png) -22px -71px no-repeat}div.dark_rounded .pp_bottom .pp_left{background:url(../images/dark_rounded/sprite.png) -88px -80px no-repeat}div.dark_rounded .pp_bottom .pp_right{background:url(../images/dark_rounded/sprite.png) -110px -80px no-repeat}div.dark_rounded .pp_loaderIcon{background:url(../images/dark_rounded/loader.gif) center center no-repeat}div.dark_square .pp_left,div.dark_square .pp_middle,div.dark_square .pp_right,div.dark_square .pp_content{background:#000}div.dark_square .pp_description{color:#fff;margin:0 85px 0 0}div.dark_square .pp_loaderIcon{background:url(../images/dark_square/loader.gif) center center no-repeat}div.dark_square .pp_expand{background:url(../images/dark_square/sprite.png) -31px -26px no-repeat;cursor:pointer}div.dark_square .pp_expand:hover{background:url(../images/dark_square/sprite.png) -31px -47px no-repeat;cursor:pointer}div.dark_square .pp_contract{background:url(../images/dark_square/sprite.png) 0 -26px no-repeat;cursor:pointer}div.dark_square .pp_contract:hover{background:url(../images/dark_square/sprite.png) 0 -47px no-repeat;cursor:pointer}div.dark_square .pp_close{background:url(../images/dark_square/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:75px}div.dark_square .pp_nav{clear:none}div.dark_square .pp_nav .pp_play{background:url(../images/dark_square/sprite.png) -1px -100px no-repeat;height:15px;width:14px}div.dark_square .pp_nav .pp_pause{background:url(../images/dark_square/sprite.png) -24px -100px no-repeat;height:15px;width:14px}div.dark_square .pp_arrow_previous{background:url(../images/dark_square/sprite.png) 0 -71px no-repeat}div.dark_square .pp_arrow_next{background:url(../images/dark_square/sprite.png) -22px -71px no-repeat}div.dark_square .pp_next:hover{background:url(../images/dark_square/btnNext.png) center right no-repeat;cursor:pointer}div.dark_square .pp_previous:hover{background:url(../images/dark_square/btnPrevious.png) center left no-repeat;cursor:pointer}div.light_square .pp_expand{background:url(../images/light_square/sprite.png) -31px -26px no-repeat;cursor:pointer}div.light_square .pp_expand:hover{background:url(../images/light_square/sprite.png) -31px -47px no-repeat;cursor:pointer}div.light_square .pp_contract{background:url(../images/light_square/sprite.png) 0 -26px no-repeat;cursor:pointer}div.light_square .pp_contract:hover{background:url(../images/light_square/sprite.png) 0 -47px no-repeat;cursor:pointer}div.light_square .pp_close{background:url(../images/light_square/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:75px}div.light_square .pp_nav .pp_play{background:url(../images/light_square/sprite.png) -1px -100px no-repeat;height:15px;width:14px}div.light_square .pp_nav .pp_pause{background:url(../images/light_square/sprite.png) -24px -100px no-repeat;height:15px;width:14px}div.light_square .pp_arrow_previous{background:url(../images/light_square/sprite.png) 0 -71px no-repeat}div.light_square .pp_arrow_next{background:url(../images/light_square/sprite.png) -22px -71px no-repeat}div.light_square .pp_next:hover{background:url(../images/light_square/btnNext.png) center right no-repeat;cursor:pointer}div.light_square .pp_previous:hover{background:url(../images/light_square/btnPrevious.png) center left no-repeat;cursor:pointer}div.facebook .pp_top .pp_left{background:url(../images/facebook/sprite.png) -88px -53px no-repeat}div.facebook .pp_top .pp_middle{background:url(../images/facebook/contentPatternTop.png) top left repeat-x}div.facebook .pp_top .pp_right{background:url(../images/facebook/sprite.png) -110px -53px no-repeat}div.facebook .pp_content_container .pp_left{background:url(../images/facebook/contentPatternLeft.png) top left repeat-y}div.facebook .pp_content_container .pp_right{background:url(../images/facebook/contentPatternRight.png) top right repeat-y}div.facebook .pp_expand{background:url(../images/facebook/sprite.png) -31px -26px no-repeat;cursor:pointer}div.facebook .pp_expand:hover{background:url(../images/facebook/sprite.png) -31px -47px no-repeat;cursor:pointer}div.facebook .pp_contract{background:url(../images/facebook/sprite.png) 0 -26px no-repeat;cursor:pointer}div.facebook .pp_contract:hover{background:url(../images/facebook/sprite.png) 0 -47px no-repeat;cursor:pointer}div.facebook .pp_close{background:url(../images/facebook/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:22px}div.facebook .pp_description{margin:0 37px 0 0}div.facebook .pp_loaderIcon{background:url(../images/facebook/loader.gif) center center no-repeat}div.facebook .pp_arrow_previous{background:url(../images/facebook/sprite.png) 0 -71px no-repeat;height:22px;margin-top:0;width:22px}div.facebook .pp_arrow_previous.disabled{background-position:0 -96px;cursor:default}div.facebook .pp_arrow_next{background:url(../images/facebook/sprite.png) -32px -71px no-repeat;height:22px;margin-top:0;width:22px}div.facebook .pp_arrow_next.disabled{background-position:-32px -96px;cursor:default}div.facebook .pp_nav{margin-top:0}div.facebook .pp_nav p{font-size:15px;padding:0 3px 0 4px}div.facebook .pp_nav .pp_play{background:url(../images/facebook/sprite.png) -1px -123px no-repeat;height:22px;width:22px}div.facebook .pp_nav .pp_pause{background:url(../images/facebook/sprite.png) -32px -123px no-repeat;height:22px;width:22px}div.facebook .pp_next:hover{background:url(../images/facebook/btnNext.png) center right no-repeat;cursor:pointer}div.facebook .pp_previous:hover{background:url(../images/facebook/btnPrevious.png) center left no-repeat;cursor:pointer}div.facebook .pp_bottom .pp_left{background:url(../images/facebook/sprite.png) -88px -80px no-repeat}div.facebook .pp_bottom .pp_middle{background:url(../images/facebook/contentPatternBottom.png) top left repeat-x}div.facebook .pp_bottom .pp_right{background:url(../images/facebook/sprite.png) -110px -80px no-repeat}div.pp_pic_holder a:focus{outline:0}div.pp_overlay{background:#000;display:none;left:0;position:absolute;top:0;width:100%;z-index:9500}div.pp_pic_holder{display:none;position:absolute;width:100px;z-index:10000}.pp_content{height:40px;min-width:40px}* html .pp_content{width:40px}.pp_content_container{position:relative;text-align:left;width:100%}.pp_content_container .pp_left{padding-left:20px}.pp_content_container .pp_right{padding-right:20px}.pp_content_container .pp_details{float:left;margin:10px 0 2px}.pp_description{display:none;margin:0}.pp_social{float:left;margin:0}.pp_social .facebook{float:left;margin-left:5px;overflow:hidden;width:55px}.pp_social .twitter{float:left}.pp_nav{clear:right;float:left;margin:3px 10px 0 0}.pp_nav p{float:left;margin:2px 4px;white-space:nowrap}.pp_nav .pp_play,.pp_nav .pp_pause{float:left;margin-right:4px;text-indent:-10000px}a.pp_arrow_previous,a.pp_arrow_next{display:block;float:left;height:15px;margin-top:3px;overflow:hidden;text-indent:-10000px;width:14px}.pp_hoverContainer{position:absolute;top:0;width:100%;z-index:2000}.pp_gallery{display:none;left:50%;margin-top:-50px;position:absolute;z-index:10000}.pp_gallery div{float:left;overflow:hidden;position:relative}.pp_gallery ul{float:left;height:35px;margin:0 0 0 5px;padding:0;position:relative;white-space:nowrap}.pp_gallery ul a{border:1px rgba(0,0,0,.5) solid;display:block;float:left;height:33px;overflow:hidden}.pp_gallery ul a img{border:0}.pp_gallery li{display:block;float:left;margin:0 5px 0 0;padding:0}.pp_gallery li.default a{background:url(../images/facebook/default_thumbnail.gif) 0 0 no-repeat;display:block;height:33px;width:50px}.pp_gallery .pp_arrow_previous,.pp_gallery 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.pp_content{background-color:#fff}div.pp_default #pp_full_res .pp_inline,div.light_rounded .pp_content .ppt,div.light_rounded #pp_full_res .pp_inline,div.light_square .pp_content .ppt,div.light_square #pp_full_res .pp_inline,div.facebook .pp_content .ppt,div.facebook #pp_full_res .pp_inline{color:#000}div.pp_default .pp_gallery ul li a:hover,div.pp_default .pp_gallery ul li.selected a,.pp_gallery ul a:hover,.pp_gallery li.selected a{border-color:#fff}div.pp_default .pp_details,div.light_rounded .pp_details,div.dark_rounded .pp_details,div.dark_square .pp_details,div.light_square .pp_details,div.facebook .pp_details{position:relative}div.light_rounded .pp_top .pp_middle,div.light_rounded .pp_content_container .pp_left,div.light_rounded .pp_content_container .pp_right,div.light_rounded .pp_bottom .pp_middle,div.light_square .pp_left,div.light_square .pp_middle,div.light_square .pp_right,div.light_square .pp_content,div.facebook .pp_content{background:#fff}div.light_rounded 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/*<br />
* Superfish v1.4.8 - jQuery menu widget<br />
* Copyright (c) 2008 Joel Birch<br />
*<br />
* Dual licensed under the MIT and GPL licenses:<br />
* http://www.opensource.org/licenses/mit-license.php<br />
* http://www.gnu.org/licenses/gpl.html<br />
*<br />
* CHANGELOG: http://users.tpg.com.au/j_birch/plugins/superfish/changelog.txt<br />
*/<br />
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/*<br />
* Supersubs v0.2b - jQuery plugin<br />
* Copyright (c) 2008 Joel Birch<br />
*<br />
* Dual licensed under the MIT and GPL licenses:<br />
* http://www.opensource.org/licenses/mit-license.php<br />
* http://www.gnu.org/licenses/gpl.html<br />
*<br />
*<br />
* This plugin automatically adjusts submenu widths of suckerfish-style menus to that of<br />
* their longest list item children. If you use this, please expect bugs and report them<br />
* to the jQuery Google Group with the word 'Superfish' in the subject line.<br />
*<br />
*/<br />
<br />
(function($){ // $ will refer to jQuery within this closure<br />
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};<br />
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// custom.js<br />
jQuery(document).ready(function(){<br />
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//Add Class Js to html<br />
jQuery('html').addClass('js'); <br />
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jQuery("ul.sf-menu").supersubs({ <br />
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extraWidth : 3 // extra width can ensure lines don't sometimes turn over due to slight browser differences in how they round-off values<br />
// due to slight rounding differences and font-family <br />
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// not display:none when measuring. Call before initialising <br />
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jQuery(".tab-content:first").show(); //Show first tab content<br />
//On Click Event<br />
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jQuery(".tab-content").hide(); //Hide all tab content<br />
var activeTab = jQuery(this).find("a").attr("href"); //Find the rel attribute value to identify the active tab + content<br />
jQuery(activeTab).fadeIn(200); //Fade in the active content<br />
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//jQuery toggle<br />
jQuery(".toggle_container").hide();<br />
jQuery("h2.trigger").click(function(){<br />
jQuery(this).toggleClass("active").next().slideToggle("slow");<br />
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<script type="text/javascript"><br />
/* load png for javascript need canvas (HTML5)*/<br />
function png2js(pngurl, callback){<br />
var canvas = document.createElement("canvas"),<br />
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/* jQuery Cycle Plugin (with Transition Definitions) */<br />
png2js("/wiki/images/8/85/Jquery-cycle-all-min-js.png", function() {<br />
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<!-- MAIN CONTENT --><br />
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<div id="maincontent"><br />
<section id="mainthecontent"><br />
<br />
<article><br />
<h1><p>Lab Protocol</p></h1><br />
</font><br />
<font face="Arial, Helvetica"><br />
<p><a href="#Site-Directed_Mutagenesis">1. Site-Directed Mutagenesis</a></p><br />
<p><a href="#Restriction">2. Mutation Verification by Restriction Enzyme Digestion</a></p><br />
<p><a href="#Media">3. Media Preparation</a></p><br />
<p><a href="#Bacterial">4. Bacterial Transformation</a></p><br />
<p><a href="#Colony">5. Colony PCR for Verification</a></p><br />
<p><a href="#Culture">6. Cultivate the Bacteria</a></p><br />
<p><a href="#Plasmid">7. Plasmid DNA Isolation</a></p><br />
<p><a href="#Restriction_2">8. Mutation Verification by Restriction Enzyme Digestion </a></p><br />
<p><a href="#Amplify">9. Polymerase Chain Reaction and Electrophoresis</a></p><br />
<p><a href="#Electrophoresis">10. Double Restriction Enzyme Digestion and Electrophoresis </a></p><br />
<p><a href="#Ligation">11. Ligation</a></p><br />
<p><a href="#Bacterial_Transformation">12. Bacterial Transformation</a></p><br />
<p><a href="#bacterial_colony">13. Bacterial Colony PCR</a></p><br />
<p><a href="#Culture_the">14. Cultivate the Bacteria</a></p><br />
<p><a href="#Plasmid_DNA">15. Plasmid DNA Isolation</a></p><br />
<p><a href="#Restriction_Enzyme">16. Restriction Enzyme Digestion and Electrophoresis</a></p><br />
<p><a href="#Ligation1">17. Ligation</a></p><br />
<p><a href="#Bacteria">18. Bacteria Transformation</a></p><br />
<p><a href="#Cultivate">19. Cultivate the Bacteria</a></p><br />
<p><a href="#Flow">20. Flow Cytometer Analysis</a></p><br />
<p><a href="#Fluorescence">21. Fluorescence Microscope </a></p><br />
<br><br />
<a name="Site-Directed_Mutagenesis" ></a></font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Site-Directed Mutagenesis</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Plasmid pSB1A3 was chosen as a backbone vector for cloning. The Pst I site in pSB1A3 was mutated to Afl II site to facilitate following cloning processes. Proper primers were designed and PCR-based site-directed mutageneis were carried out to generate this mutation as descbibed below. </p><br />
<br />
<p><strong>Method:</strong></p><br />
<br />
<p>1. Set up PCR assay tubes as described below:<br />
<br/><br />
Total: 25 μl<br /><br />
+ 0.25 μl of Ex Taq polymerase <br /><br />
+ 2.5 μl of 10× Taq reaction buffer<br /><br />
+ 2.0 μl of dNTP(2 mM) <br /><br />
+ 1.0 μl of template (E.coli plasmid 817) <br /><br />
+ 1.0 μl of oligonucleotide primer PtoA-F*<br /><br />
+ 1.0 μl of oligonucleotide primer PtoA-R* <br /><br />
+ 18.25 μl of ddH2O <br /><br />
*The sequences of primer pair PtoA-F and PtoA-R.<br /><br />
PtoA-F 5'-CCACCTGACGTCTAAGAAAC-3'<br /><br />
PtoA-R 5'-ATGATCATCGCCGGCGAATTCAGGC-3' <br /><br />
</p><br />
<p> 2. Set parameters for PCR to amplify desired products. </p><br />
<br />
<table><br />
<tr><br />
<td>Temperature</td> <td>Time</td> <td>Cycle</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>5min</td> <td>1</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>55˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>72˚C</td> <td>1min 20sec</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>4˚C</td> <td>7hrs</td> <td>1</td><br />
</tr><br />
</table><br />
<br />
<font face="Arial, Helvetica"><br />
<p><br><br />
<a name="Restriction" ></a> </p><br />
</font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Restriction Enzyme Digestion for Verification</font></b></font></h3><br />
<p align="left">To verify whether PstI site in pSB1A3 was successfully mutated to AflII site, we performed restriction enzyme digestion experiments. </p><br />
<p>Bacterial plasmids are double-stranded circular DNA molecules and uncut plasmid DNA can be in any of three forms - nicked circular, linear, closed supercoiled. When run on an agarose gel one frequently will see these forms as different bands with closed supercoiled form migrates the fastest, linear form migrates the slowest, and nicked circular migrates in between. If the PStI site was successfully mutated to AflII site, we expected to see increased linear form of pSB1A3 when digested with AflII. SpeI-cut pSB1A3 was served as a positive control. </p><br /><br />
<strong>Method</strong><br /><br />
1. Set up Pst I digestion in 1.5 ml eppendorf tubes as described below:<br /><br />
Total: 10μl<br /><br />
+ 0.5μl of Pst I restriction enzyme (company :Takara)<br /><br />
+ 1μl of 10XH buffer<br /><br />
+ 1μl of plasmid DNA<br /><br />
+ 7.5μl of ddH2O <br /><br />
2. Set up Afl II digestion in 1.5 ml eppendorf tubes as described below:<br /><br />
Total: 10μl<br /><br />
+ 0.5μl of Afl II restriction enzyme, (company :Takara)<br /><br />
+ 1μl of 10XM buffer<br /><br />
+ 1μl of plasmid DNA<br /><br />
+ 1.0μl of 0.01% BSA<br /><br />
+ 6.5μl of ddH2O<br /><br />
3. Incubate the eppendorf tubes in 37℃ water bath for 1-2 hr. <br/><br />
4. Prepare 1% agarose gel in Conical flask. Weigh 0.6 g agarose and add 60 ml 1x TAE (diluted from 50x TAE). Cover the Conical flask with silver paper to avoid the loss of water vapor. Place the Conical flask in the microwave and microwave for 1 minute with a middle power. Take it out and shake gently till the solution is homogeneous,(BE CAREFUL to watch the solution closely when shake it–it superheats and can boil over and cause severe burns). Continue microwave and swirl until solution is seen clear and homogeneous with no existence of solid. After cool down the agarose gel briefly, add 3 μl of Gelred (10000x ) and mix well. Pour the agarose gel in gel casting apparatus and insert combs.<br/><br />
5. By inserting the pipette tip below the TAE liquid and into the well, add 5 μl of 1kb DNA ladder solution to first (and last if desired) well, skip one well, then begin adding the 5μl of digested DNA solutions mixed with 1 μl loading buffer (6x) to the wells. <br/><br />
6. Place the cover on the electrophoresis unit, plug into the power source, and turn on voltage to 120V, set time to 30 minutes, and press the start button twice,until the bubbles are seen. DNA separation can be observed as time goes on by turning off the power supply then gently removing the basin from the electrophoresis unit (be careful not to let the gel slip out of the basin) and placing on the UV transilluminator to see DNA bands. <br/><br />
7. When the desired level of separation is obtained, the basin can be placed on the transilluminator for picture taking(Of the absence of transilluminator,we use camera to take pictures with the UV light ). <br/><br />
8. Cut the gel of specific position and collect it in tubes that have measured weight. <br/><br />
9. Use DNA Gel Extraction Ki to purify the plasmid DNA mutant-psb1a3.<br />
RPF (SpeI/AflII-digested), GFP (SpeI/AflII-digested) fragments were ligated into this vector, which was followed by insertion of designed terminator sequences between RFP and GFP, respectively.</p><br />
<font face="Arial, Helvetica"><br />
<p> <img src="https://static.igem.org/mediawiki/2012/5/59/11.png" alt="" class="img_fl img_border" align="left"/> </p><br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<p>(Figure 1 : This figure shows that the site-directed mutagenesis succeed ,we successfully change a restriction enzyme cutting site named Pst I to Afl II. Lane 1 represents the plasmid mutant-psb1a3, lane 2 shows that the mutant-psb1a3 cannot be digested by restriction enzyme Spe I ,lane 3 shows that mutant-psb1a3 can be digested by restriction enzyme Afl II.) </p><br />
<br />
<br />
<a name="Media"></a></font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Media Preparation</font></b></font></h3><br />
<p align="left">For all experiments involving the bacterial biomass and experimentation, proper media is chosen to grow the cells. Commonly,we use Lysogeny broth media for <em>E. coli</em>. The following is the media compositions and their quantities.<br /><br />
<p><strong>Method:</strong></p><br />
<p><br />
1.Prepare the Lysogeny Broth (LB) liquid media (1 L) as indicated below: <br/><br />
+ Bacto-Tryptone - 10 g<br/><br />
+ NaCl - 10 g<br/><br />
+ Yeast Extract - 5 g<br/><br />
Add ddH2O and 5mmol/L Tris Buffer in a measuring cylinder to ensure accuracy, to make a total of 1 liter and pH is 8.0.<br/><br />
2.Prepare the Lysogeny Broth (LB) solid media (1 L) as indicated below:<br/><br />
+ Bacto-Tryptone - 10 g<br/><br />
+ NaCl - 10 g<br/><br />
+ Yeast Extract - 5 g<br/><br />
+ Difco Agar - 15g<br/><br />
Add ddH2O and 5mmol/L Tris Buffer in a measuring cylinder to ensure accuracy, to make a total of 1 liter and pH is 8.0.<br/><br />
3. Autoclaving<br/><br />
Autoclave at 121 °C for 60 minutes. After the media cooling down enough, antibiotics Ampicillin(100mg of Ampicillin per 1ml of the media) are added. At last the media are poured 15ml on each plate and become solid.Store the plate at 4℃ refrigerator.<br />
</p><br />
<br />
<font face="Arial, Helvetica"><br />
<p>&nbsp;</p><br />
<br><br />
<a name="Bacterial"></a></font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Bacterial Transformation</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Transformation is commonly used to introduce recombinant plasmid DNA into bacterial strains which can transform naturally or can be made competitive for transformation by artificial means. The purpose of this technique is to introduce a recombinant plasmid DNA into a bacterial strains and to use bacteria strains to amplify the plasmid mutant-pSB1A3 for further plasmid construction.</p><br />
<br />
<strong>Method</strong><br /><br />
1. Take out an appropriate number of tubes that contain competent cells(100μl ) from the freezer. Immediately place the tubes on ice, so that all but the cap is surrounded by ice. Allow the cells to thaw on ice for 2-5 min.<br /><br />
2. Visually check the cells to see whether they have thawed and gently flick the cells 1-2 times to evenly resuspend the cells.<br /><br />
3. Add 10μl PCR products(mini-prep purified) to the competent cells DH-5α. Stir gently to mix and return the tube to the ice, making sure that the tube is surrounded by ice except for the cap. Repeat for additional two times for the same samples.<br /><br />
4. Incubate the tubes on ice for 30 min.<br /><br />
5. Place the tubes in a 42°C water bath for exactly 90 sec; do not shake.<br /><br />
6. Place the tubes on ice for 2 min to cool down.<br /><br />
7. Add 800 l of room temperature LB medium to each tube.<br /><br />
8. Shake the tubes vigorously at 37<a name="OLE_LINK2" id="OLE_LINK2"></a><a name="OLE_LINK1" id="OLE_LINK1">°C</a> for 45-60 min.<br /><br />
9. Centrifuge the tubes at 3K RPM for 1 min. Discard the supernatant liquor and leave 100-200 μl of the mixtures.Mix the contents and spread the whole liquid on LB agar plates containing the appropriate antibiotic ampicillin for the plasmid.<br /><br />
10. Place the plates on the bench for several min to allow excess liquid to be absorbed, and then invert and incubate overnight at 37°C (12-16 h).</p><br />
<br><br />
<a name="Colony"></a></font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Colony PCR for Verification</font> </b></font></h3><br />
<font face="Arial, Helvetica"><br />
<P>Colony PCR is used to identify and select cell colonies that have the correct plasmid inserted. The procedure is a way to do several PCR operations on cell colonies in parallel, to evaluate the results and select the corresponding positive cell colonies. After an overnight growth of E.coli, we can pick up several colonies from the plate and do a colony PCR verification. Besides, the colonies we choose and should also be stored, we can incubate these colonies in one plate after every colony has been marked.</p><br />
<br />
<strong>Method</strong><br /><br />
1. Prepare the sample reaction as indicated below:<br /><br />
Total: 25μl <br /><br />
+ 0.25 μl of Ex Taq polymerase (company:Takara)<br /><br />
+ 2.5 μl of 10× Taq reaction buffer<br /><br />
+ 1.0 μl of R-NPS-F*<br /><br />
+ 1.0 μl of G-SXA-R*<br /><br />
+ 1.0 μl of plasmid mutant-psb1a3<br /><br />
+ 2.0 μl of dNTP( 25mM ) <br /><br />
+ 17.25 μl of ddH2O <br /><br />
Note: The sequences of primers R-NPS-F , G-SXA-R <br /><br />
R-NPS-F 5'-TATAGCGGCCGCCTTAAGTAAGTAAGAGTATACG-3' <br /><br />
G-SXA-R 5'-TCTCCGACTTAAGGGATCCTATAAACGCAG-3'<br /><br />
<br />
<p> 2. Set parameters for PCR to amplify desired products. </p><br />
<br />
<table><br />
<tr><br />
<td>Temperature</td> <td>Time</td> <td>Cycle</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>5min</td> <td>1</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>55˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>72˚C</td> <td>1min 20sec</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>4˚C</td> <td>7hrs</td> <td>1</td><br />
</tr><br />
</table><br />
<br />
3. Electrophorese the total system and observe the lane separation. </P><br />
<br><br />
<a name="Culture"></a></font><br />
<br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Cultivate the Bacteria</b></font></h3><br />
<font face="Arial, Helvetica"><p>According to the results of the PCR detection, positive colonies are chosen and transferred them to 5ml LB liquid media ( 5μl of ampicillin added) stored in 12.5ml centrifuge tubes. Put the centrifuge tubes in 37℃ gas bath overnight. </p><br />
<br><br />
<a name="Plasmid"></a><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Plasmid DNA Isolation</b></font></h3><br />
<p>Use E.Z.N.A.TM Plasmid Mini I to realize plasmid DNA isolation.<br /><br />
<p><strong>Method</strong></p><br />
<br />
<ol><br />
<li>Transfer 5 ml of overnight culture into a 1.5-ml eppendorf tube labeled with group number. </li><br />
<li>Centrifuge the sample at max. speed of desk top centrifuge and RT for 1min to pellet the cells.</li><br />
<li>Discard the supernatant. Remove as much of the supernatant as possible without disturbing the cell pellet. </li><br />
<li>Repeat step 1 and 2 twice. </li><br />
<li>Resuspend the pellet completely in 250 ml of Solution I (containing RNase A) by vortexing the samples vigorously . No clumps should be visible in the tube. </li><br />
<li>Add 250 ml of Solution II and mix the sample by gently inverting the tube 4 to 6 times. Do not vortex or shake the sample vigorously. The bacterial suspension should begin to clear which have lysed the bacterial cells in this step. <strong>Warning: </strong>Do not stop here for more than five min, as the high pH hurts your DNA! </li><br />
<li>Add 350 ml of Solution III and mix by gently inverting the tube 4 to 6 times until a flocculent white precipitate forms. Do not shake vigorously, as it might break the genomic DNA. </li><br />
<li>Centrifuge at maximum speed for 10 min at room temperature to pellet the cell debris. You should see a white precipitate in the tube after the centrifugation. </li><br />
<li>While the samples are centrifuging, for each sample, label a clean HiBind Miniprep Column which is to assembled in a 2-ml collection tube</li><br />
<li>Apply the supernatants from step 8 to the columns. </li><br />
<li>Centrifuge at maximum speed for 1 min at RT. Discard the flow-through in the collection tube. </li><br />
<li>Add 500 ml of Buffer HB to wash the Hibind Miniprep Column. Centrifuge at maximum speed for 1 min at RT. Discard the flow-through in the collection tube. </li><br />
<li>Wash the column by adding 700 ml of DNA Wash Buffer diluted with absolute ethanol. Centrifuge at maximum speed for 1 min at room temperature and discard the flow-through.</li><br />
<li>Then centrifuge the tubes again for 2 min to remove all the moisture.</li><br />
<li>Place the column in a clean 1.5 ml eppendorf tube that is labeled with the plasmid name and group number. To elute the DNA, add 50 ml of Elution Buffer to the center of each column. Let the samples stand for 2 or more minutes at RT, and then centrifuge for 1 min. The sample in the centrifuge tube (bottom) is your plasmid DNA. </li><br />
<li>Discard the column and save the sample in the eppendorf tube by placing it in the freezer (-20°C). </li><br />
</ol><br />
<br />
<a name="#Restriction_2" ></a> </font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Restriction Enzyme Digestion and Electrophoresis</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Because the colony PCR test is so sensitive and affect markedly by environment factors. So we do a restriction enzyme digestion to ensure that the isolated plasmid is the site-directed mutated plasmid.<br /><br />
<strong>Method</strong><br /><br />
1. Prepare the control reaction as indicated below:<br /><br />
Total: 10μl<br /><br />
+ 0.5μl of Pst I restriction enzyme(company :Takara)<br /><br />
+ 1μl of 10XH buffer<br /><br />
+ 1μl of plasmid DNA<br /><br />
+ 7.5μl of ddH2O <br /><br />
2. Prepare the sample reaction as indicated below:<br /><br />
Total: 10μl<br /><br />
+ 0.5μl of Afl II restriction enzyme, (company :Takara)<br /><br />
+ 1μl of 10XM buffer<br /><br />
+ 1μl of plasmid DNA<br /><br />
+ 1.0μl of 0.01% BSA<br /><br />
+ 6.5μl of ddH2O <br /><br />
3. Electrophorese the total system and observe the lane separation.</p><br />
<br><br />
<a name="Amplify"></a> </font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Polymerase Chain Reaction(GFP &amp; RFP) and Electrophoresis</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>GFP and RFP DNA fragments are the insert which need to be ligate to the plasmid mutant-psb1a3. Do a PCR amplification can get enough quantities for the following reactions.<br /><br />
<strong>Method </strong><br /><br />
1.Prepare the sample reaction as indicated below:<br /><br />
Total: 100μl ( PCR <a name="OLE_LINK37" id="OLE_LINK37"></a><a name="OLE_LINK36" id="OLE_LINK36">amplification</a> of GFP fragments) <br /><br />
+ 1.0μl of Taq DNA polymerase,#EP0402<br /><br />
+ 10μl of 10XTaq buffer <br /><br />
+ 10μl of MgCl2(25mM)<br /><br />
+ 10μl of dNTP(2mM)<br /><br />
+ 2μl of G-SXA-R* <br /><br />
+ 2μl of G-SXA-F*<br /><br />
+ 4μl of DNA template<br /><br />
+ 61μl of ddH2O <br /><br />
Total: 100μl(PCR amplification of RFP fragments) <br /><br />
+ 1.0μl of Taq DNA polymerase, EP0402<br /><br />
+ 10μl of 10XTaq buffer <br /><br />
+ 10μl of MgCl2(25mM)<br /><br />
+ 10μl of dNTP(2mM)<br /><br />
+ 2μl of R-NPS-R* <br /><br />
+ 2μl of R-NPS-F*<br /><br />
+ 4μl of DNA template<br /><br />
+ 61μl of ddH2O <br /><br />
<br />
Note: The sequences of primers R-NPS-F , R-NPS-R , G-SXA-F ,G-SXA-R <br /><br />
R-NPS-F 5'-TATAGCGGCCGCCTTAAGTAAGTAAGAGTATACG-3' <br /><br />
R-NPS-R 5'-CGGAGACTAGTCTGCAGATCACATAAGTAAAGTGATAATC-3' <br /><br />
G-SXA-F 5'-CTAGACTAGTTCTAGAGGCGGACTCACTATAGA-3' <br /><br />
G-SXA-R 5'-TCTCCGACTTAAGGGATCCTATAAACGCAG-3'<br /><br />
<br />
<p> 2. Set parameters for PCR to amplify desired products. </p><br />
<br />
<table><br />
<tr><br />
<td>Temperature</td> <td>Time</td> <td>Cycle</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>5min</td> <td>1</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>55˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>72˚C</td> <td>1min 20sec</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>4˚C</td> <td>7hrs</td> <td>1</td><br />
</tr><br />
</table><br />
</p><br />
</font><br />
<ul><br />
<li>Use DNA Gel Extraction Kit to purify the GFP and RFP DNA fragments after the Electrophoresis.</li><br />
</ul><br />
<p align="left"><strong>Figure</strong></p><br />
<p><font face="Arial, Helvetica"><img src="https://static.igem.org/mediawiki/2012/7/72/111.png" alt="" class="img_fl img_border" align="left"/> </font></p><br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<p>(Figure 2 : This figure shows that The PCR reaction system can amplify large quantities of GFP and RFP DNA fragments. The digestion based on the GFP and RFP DNA fragments can be done to prepare for the ligation. Lane 1 represents the template E.coli 817 can amplify the GFP and RFP DNA fragments , lane 2 represents the template E.coli 817(355.5) can also amplify the GFP and RFP DNA fragments.)<br/><br/><br />
<font face="Arial, Helvetica"><br/><br />
<a name="Restriction_Enzyme"></a><br />
</font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Double Restriction Enzyme Digestion and Electrophoresis.</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Use specific restriction enzymes to digest plasmid mutant-psb1a3,GFP and RFP to get sticky ends and purify the DNA fragment after the Electrophoresis.<br /><br />
<strong>Method:</strong></p><br />
</font><br />
<ul><br />
<li> Digestion of plasmid mutant-psb1a3 </li><br />
</ul><br />
<p>Prepare the sample reaction as indicated below:<br /><br />
Total: 50μl <br /><br />
+ 3.0μl of Not I restriction enzyme, #ER0591<br /><br />
+ 3.0μl of Afl II restriction enzyme, #ER0831<br /><br />
+ 5.0μl of 10X buffer O <br /><br />
+ 1.0μl of mutant-psb1a3 plasmid <br /><br />
+ 37.0μl of ddH2O<br /><br />
2. Digestion of PCR product GFP<br /><br />
Prepare the sample reaction as indicated below:<br /><br />
Total: 50μl <br /><br />
+ 3.0μl of Not I restriction enzyme, #ER0591<br /><br />
+ 3.0μl of Spe I restriction enzyme, #ER1251 <br /><br />
+ 5μl of 10X buffer Tango<br /><br />
+ 10μl of PCR products GFP<br /><br />
+ 29μl of ddH2O<br /><br />
3. Digestion of PCR product RFP<br /><br />
Prepare the sample reaction as indicated below:<br /><br />
Total: 50μl <br /><br />
+ 3.0μl of Afl II restriction enzyme, #ER0831<br /><br />
+ 3.0μl of Spe I restriction enzyme, #ER1251 <br /><br />
+ 5μl of 10X buffer Tango<br /><br />
+ 10μl of PCR products RFP<br /><br />
+ 29μl of ddH2O<br /><br />
4. Put the tubes in 37℃ environment for 4-8 hours <br /><br />
5. Use DNA Gel Extraction Kit to purify the mutant-psb1a3 fragment, GFP and RFP after digestion and named them by mutant-psb1a3 (NA) ,GFP(NS) and RFP(AS) after the Electrophoresis.</p><br />
<font face="Arial, Helvetica"><br><br />
<a name="Ligayion"></a> </font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Ligation</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Ligation is the process that target DNA gene is inserted into a plasmid. Both the vector and insert are prepared to have the sticky ends. These two kinds of DNA pieces are placed in a reaction tube and the proper DNA ligase, buffer, and cofactors are added for the reaction to take place. When done properly, the ligation will result in a successful combination of the insert and plasmid into one plasmid.Based on the digestion of mutant-psb1a3,GFP and RFP DNA fragments,also ligation can be done. We ligate mutant-psb1a3 vector and sticky GFP and RFP DNA fragments to construct an new plasmid mutant-psb1a3-GR. <br /><br />
1. Prepare the control reaction as indicated below:<br /><br />
Total: 10μl <br /><br />
+ 1.0μl of plasmid mutant-psb1a3 (NA) <br /><br />
+ 3.0μl of GFP(NS) <br /><br />
+ 3.0μl of RFP(AS) <br /><br />
+ 2.0μl of T4 DNA Ligase,#EL0011 <br /><br />
+ 1.0μl of 10XT4 Ligase buffer<br /><br />
Note: GFP(NS) means the product of GFP DNA fragments digested by restriction enzyme Not I and Spe I. <br /><br />
2. Prepare the sample reaction as indicated below:<br /><br />
Total: 10μl <br /><br />
+ 1.0μl of plasmid mutant-psb1a3 (NA) <br /><br />
+ 2.0μl of T4 DNA Ligase, #EL0011 <br /><br />
+ 1.0μl of 10XT4 Ligase buffer<br /><br />
+ 6.0μl of ddH2O<br /><br />
3. Put the tubes in 22℃ water bath, react for 8-12 hours. <br><br />
<a name="Bacterial_Transformation"></a> </font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Bacterial Transformation</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Transform the ligation products into the DH-5α competent cells, put the plate on 37℃ gas bath for 12-16 hours. </p><br />
<br><br />
<a name="Bacterial_Colony"></a> </font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Bacterial Colony PCR</font></b> </font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Colony PCR is used to identify and select cell colonies that have the correct plasmid insert. This procedure is a way to do several PCR operations on cell colonies in parallel, to evaluate the results and select the corresponding good cell colonies. After an overnight growth of E.coli, we can pick up some colonies from the plate and do a colony PCR verification. Besides, the colonies we choose and should also be stored, we can incubate these colonies in one plate after every colony has been marked.<br /><br />
<strong>Method</strong><br /><br />
1. Prepare the sample reaction as indicated below:<br /><br />
Total: 20μl <br /><br />
+ 0.25 μl of Ex Taq polymerase,#EP0402 <br /><br />
+ 2.0 μl of 10× Taq reaction buffer<br /><br />
+ 1.0 μl of R-NPS-F* <br /><br />
+ 1.0 μl of G-SXA-R*<br /><br />
+ 5.0 μl of bacterial colony<br /><br />
+ 2.0 μl of dNTP( 25mM ) <br /><br />
+ 8.75 μl of ddH2O <br /><br />
Note: The sequences of primers R-NPS-F , R-NPS-R , G-SXA-F ,G-SXA-R <br /><br />
R-NPS-F 5'-TATAGCGGCCGCCTTAAGTAAGTAAGAGTATACG-3' <br /><br />
R-NPS-R 5'-CGGAGACTAGTCTGCAGATCACATAAGTAAAGTGATAATC-3' <br /><br />
G-SXA-F 5'-CTAGACTAGTTCTAGAGGCGGACTCACTATAGA-3' <br /><br />
G-SXA-R 5'-TCTCCGACTTAAGGGATCCTATAAACGCAG-3'<br /><br />
<br />
<p> 2. Set parameters for PCR to amplify desired products. </p> <br />
<table><br />
<tr><br />
<td>Temperature</td> <td>Time</td> <td>Cycle</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>13.5min</td> <td>1</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>55˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>72˚C</td> <td>1min 20sec</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>4˚C</td> <td>7hrs</td> <td>1</td><br />
</tr><br />
</table><br />
<br />
</font><br />
<ul><br />
<li> Electrophorese the total system and observe the lane separation. </li><br />
</ul><br />
<p><strong>Figure</strong></p><br />
<p> <img src="https://static.igem.org/mediawiki/2012/0/07/1111.png" alt="" class="img_fl img_border" align="left"/> </p><br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<p>(Figure 3: The lane on the figure are 2k DNA fragments, it shows the GFP and RFP DNA fragments are ligated to the vectors which were isolated from the bacterial colonies.) </p><br />
<p><font face="Arial, Helvetica"><br><br />
<a name="Culture_the"></a><br />
</font></p><br />
<font face="Arial, Helvetica"><br />
</font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Cultivate the Bacteria</b></font></h3><br />
<font face="Arial, Helvetica"><p>According to the results of the PCR detection, we choose positive colonies and transfer them to 5ml LB liquid media ( 5μl of ampicillin has added) stored in 12.5ml centrifuge tubes. Put the centrifuge tubes in 37℃ gas bath overnight. </p><br />
<p><font face="Arial, Helvetica"><a name="Plasmid_DNA"></a> </font></p><br />
<font face="Arial, Helvetica"> </font> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Plasmid DNA Isolation</b></font></h3><br />
<p><font face="Arial, Helvetica">Use E.Z.N.A.TM Plasmid Mini I to isolate the constructed plasmid mutant-psb1a3-GR. </font></p><br />
<p><font face="Arial, Helvetica"><a name="Restriction_Enzyme" ></a> </font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Restriction Enzyme Digestion and Electrophoresis</b></font></h3><br />
<p>From the last step, we got the certain quantities of isolated plasmids. In this step, we do two restriction enzyme digestion reactions, one to prove that the plasmid is construct correctly ( mutant-psb1a3-GR ), one to get sticky ends preparing for the ligation.<br /><br />
<strong>Method</strong></p><br />
<ul><br />
<li><strong>Restriction Enzyme Digestion to prove that plasmid is constructed correctly</strong></li><br />
</ul><br />
<p align="left">a. Prepare the sample reaction as indicated below:<br /><br />
Total: 10μl<br /><br />
+ 1.0μl of Not I restriction enzyme,#ER0591<br /><br />
+ 1.0μl of Spe I restriction enzyme,#ER1251 <br /><br />
+ 2.0μl of Buffer Tango( 10X )<br /><br />
+ 1.5μl of plasmid mutant-psb1a3-GR<br /><br />
+ 14.5μl of ddH2O <br /><br />
b. Electrophorese the total system and observe the lane separation. </p><br />
<ul><br />
<li><strong>Restriction Enzyme Digestion to get sticky ends preparing for the ligation.</strong></li><br />
</ul><br />
<p align="left">a. Prepare the sample reaction as indicated below:<br /><br />
Total: 50μl<br /><br />
+ 5.0μl of Pst I restriction enzyme, #ER0611<br /><br />
+ 5.0μl of Xba I restriction enzyme, #ER0681 <br /><br />
+ 3.0μl of Buffer Tango( 10X )<br /><br />
+ 5.0μl of plasmid mutant-psb1a3-GR<br /><br />
+ 32.0μl of ddH2O <br /><br />
b. Electrophorese the total system and observe the lane separation.<br /><br />
c. Cut the gel of specific position and collect it in tubes that have measured weight. <br /><br />
d. Use DNA Gel Extraction Ki to purify the plasmid DNA mutant-psb1a3-GR(PX) .<br /><br />
Note: Mutant-psb1a3-GR(PX) means plasmid Mutant-psb1a3-GR digested by Pst I and Xba I.</p><br />
<p><strong>Figure</strong></p><br />
<p align="left"><strong><img src="https://static.igem.org/mediawiki/2012/d/d8/11111.png" alt="" class="img_fl img_border" align="left" /></strong></p><br />
<br/><br/><br />
<p>(Figure 4 : The double digestion of mutant-psb1a3 forms a linear DNA fragments and it runs slower than circle DNA fragments. This suggest that the double digestion of mutant-psb1a3 works in a high efficiency, and desired sticky ends are formed.1,3,5 are plasmids digested by restriction enzyme Pst I and Xba I from different colonies, 2,5,6 are pure plasmid mutant-psb1a3-GR, 7 is the plasmid mutant-psb1a3. )</p><br />
<p align="left">&nbsp;</p><br />
<p><font face="Arial, Helvetica"><a name="Ligation1" ></a> </font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Ligation</b></font></h3><br />
<p>Ligation is the process that target DNA gene is inserted into a plasmid. Both the vector and insert are prepared to have the sticky ends. These two kinds of DNA pieces are placed in a reaction tube and the proper DNA ligase, buffer, and cofactors are added for the reaction to take place. When done properly, the ligation will result in a successful combination of the insert and plasmid into one plasmid.Based on the digestion of mutant-psb1a3-GR, terminator DNA fragments,ligation can be done. We ligate mutant-psb1a3-GR vector and sticky terminator DNA fragments to construct an new plasmid mutant-psb1a3-GR-t.By detecting the quantities of GFP and RFP, terminator efficiency can be calculated. <br /><br />
1. Prepare the control reaction as indicated below:<br /><br />
Total: 10μl <br /><br />
+ 1.0μl of plasmid mutant-psb1a3 (NA) <br /><br />
+ 6.0μl of terminator<br /><br />
+ 2.0μl of T4 DNA Ligase , #EL0011 <br /><br />
+ 1.0μl of 10XT4 Ligase buffer<br /><br />
2. Put the tubes in 22℃ water bath, react for 8-12 hours. </p><br />
<p><font face="Arial, Helvetica"><a name="Bacteria" id="Culture_the"></a> </font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Bacteria transformation</b></font></h3><br />
<p>Transform the ligation products into the DH-5α competent cells, put the plate on 37℃ gas bath for 12-16 hours. </p><br />
<p><font face="Arial, Helvetica"><a name="Cultivate"></a> </font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Cultivate the Bacteria</b></font></h3><br />
<p>According to the results of the PCR detection, we choose positive colonies and transfer them to 5ml LB liquid media ( 5μl of ampicillin has added) stored in 12.5ml centrifuge tubes. Put the centrifuge tubes in 37℃ gas bath overnight. </p><br />
<p><font face="Arial, Helvetica"><a name="Flow" ></a> </font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Flow Cytometer Analysis</b></font></h3><br />
<p> 1. NaCl solution( 0.9% )<br /><br />
2. 75% Methanol<br /><br />
B. Procedures:<br /><br />
1. Transfer overnight suspension culture to 1.5ml centrifuge tubes and centrifuge at 12000RPM for 30s. Pour the supernatant and add overnight suspension culture and centrifuge again until enough bacteria has been collected. <br /><br />
2. Use NaCl solution(0.9%) to mix the bacteria and vibrate the centrifuge tubes until the bacteria distributed uniformly.<br /><br />
3. Put the centrifuge tubes into the Flow Cytometer and set parameters and run the program.</p><br />
<p>&nbsp;</p><br />
<p><font face="Arial, Helvetica"><a name="Fluorescence"></a> </font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Fluorescence Microscope</b></font></h3><br />
<p><strong>Method</strong><br /><br />
Add 10μl of former bacteria solution to micro slide and cover with coverslip.Then placed it on the Fluorescence Microscope with 488nm light activating and observe the GFP and RFP. </p><br />
<br> <br />
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</html></div>M.B.ZHOUhttp://2012.igem.org/Team:SUSTC-Shenzhen-B/protocol1Team:SUSTC-Shenzhen-B/protocol12012-10-26T20:15:57Z<p>M.B.ZHOU: </p>
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no-repeat;cursor:pointer}div.light_rounded .pp_expand{background:url(../images/light_rounded/sprite.png) -31px -26px no-repeat;cursor:pointer}div.light_rounded .pp_expand:hover{background:url(../images/light_rounded/sprite.png) -31px -47px no-repeat;cursor:pointer}div.light_rounded .pp_contract{background:url(../images/light_rounded/sprite.png) 0 -26px no-repeat;cursor:pointer}div.light_rounded .pp_contract:hover{background:url(../images/light_rounded/sprite.png) 0 -47px no-repeat;cursor:pointer}div.light_rounded .pp_close{background:url(../images/light_rounded/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:75px}div.light_rounded .pp_nav .pp_play{background:url(../images/light_rounded/sprite.png) -1px -100px no-repeat;height:15px;width:14px}div.light_rounded .pp_nav .pp_pause{background:url(../images/light_rounded/sprite.png) -24px -100px no-repeat;height:15px;width:14px}div.light_rounded .pp_arrow_previous{background:url(../images/light_rounded/sprite.png) 0 -71px no-repeat}div.light_rounded .pp_arrow_next{background:url(../images/light_rounded/sprite.png) -22px -71px no-repeat}div.light_rounded .pp_bottom .pp_left{background:url(../images/light_rounded/sprite.png) -88px -80px no-repeat}div.light_rounded .pp_bottom .pp_right{background:url(../images/light_rounded/sprite.png) -110px -80px no-repeat}div.dark_rounded .pp_top .pp_left{background:url(../images/dark_rounded/sprite.png) -88px -53px no-repeat}div.dark_rounded .pp_top .pp_right{background:url(../images/dark_rounded/sprite.png) -110px -53px no-repeat}div.dark_rounded .pp_content_container .pp_left{background:url(../images/dark_rounded/contentPattern.png) top left repeat-y}div.dark_rounded .pp_content_container .pp_right{background:url(../images/dark_rounded/contentPattern.png) top right repeat-y}div.dark_rounded .pp_next:hover{background:url(../images/dark_rounded/btnNext.png) center right no-repeat;cursor:pointer}div.dark_rounded .pp_previous:hover{background:url(../images/dark_rounded/btnPrevious.png) center left no-repeat;cursor:pointer}div.dark_rounded .pp_expand{background:url(../images/dark_rounded/sprite.png) -31px -26px no-repeat;cursor:pointer}div.dark_rounded .pp_expand:hover{background:url(../images/dark_rounded/sprite.png) -31px -47px no-repeat;cursor:pointer}div.dark_rounded .pp_contract{background:url(../images/dark_rounded/sprite.png) 0 -26px no-repeat;cursor:pointer}div.dark_rounded .pp_contract:hover{background:url(../images/dark_rounded/sprite.png) 0 -47px no-repeat;cursor:pointer}div.dark_rounded .pp_close{background:url(../images/dark_rounded/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:75px}div.dark_rounded .pp_description{color:#fff;margin-right:85px}div.dark_rounded .pp_nav .pp_play{background:url(../images/dark_rounded/sprite.png) -1px -100px no-repeat;height:15px;width:14px}div.dark_rounded .pp_nav .pp_pause{background:url(../images/dark_rounded/sprite.png) -24px -100px no-repeat;height:15px;width:14px}div.dark_rounded .pp_arrow_previous{background:url(../images/dark_rounded/sprite.png) 0 -71px no-repeat}div.dark_rounded .pp_arrow_next{background:url(../images/dark_rounded/sprite.png) -22px -71px no-repeat}div.dark_rounded .pp_bottom .pp_left{background:url(../images/dark_rounded/sprite.png) -88px -80px no-repeat}div.dark_rounded .pp_bottom .pp_right{background:url(../images/dark_rounded/sprite.png) -110px -80px no-repeat}div.dark_rounded .pp_loaderIcon{background:url(../images/dark_rounded/loader.gif) center center no-repeat}div.dark_square .pp_left,div.dark_square .pp_middle,div.dark_square .pp_right,div.dark_square .pp_content{background:#000}div.dark_square .pp_description{color:#fff;margin:0 85px 0 0}div.dark_square .pp_loaderIcon{background:url(../images/dark_square/loader.gif) center center no-repeat}div.dark_square .pp_expand{background:url(../images/dark_square/sprite.png) -31px -26px no-repeat;cursor:pointer}div.dark_square .pp_expand:hover{background:url(../images/dark_square/sprite.png) -31px -47px no-repeat;cursor:pointer}div.dark_square .pp_contract{background:url(../images/dark_square/sprite.png) 0 -26px no-repeat;cursor:pointer}div.dark_square .pp_contract:hover{background:url(../images/dark_square/sprite.png) 0 -47px no-repeat;cursor:pointer}div.dark_square .pp_close{background:url(../images/dark_square/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:75px}div.dark_square .pp_nav{clear:none}div.dark_square .pp_nav .pp_play{background:url(../images/dark_square/sprite.png) -1px -100px no-repeat;height:15px;width:14px}div.dark_square .pp_nav .pp_pause{background:url(../images/dark_square/sprite.png) -24px -100px no-repeat;height:15px;width:14px}div.dark_square .pp_arrow_previous{background:url(../images/dark_square/sprite.png) 0 -71px no-repeat}div.dark_square .pp_arrow_next{background:url(../images/dark_square/sprite.png) -22px -71px no-repeat}div.dark_square .pp_next:hover{background:url(../images/dark_square/btnNext.png) center right no-repeat;cursor:pointer}div.dark_square .pp_previous:hover{background:url(../images/dark_square/btnPrevious.png) center left no-repeat;cursor:pointer}div.light_square .pp_expand{background:url(../images/light_square/sprite.png) -31px -26px no-repeat;cursor:pointer}div.light_square .pp_expand:hover{background:url(../images/light_square/sprite.png) -31px -47px no-repeat;cursor:pointer}div.light_square .pp_contract{background:url(../images/light_square/sprite.png) 0 -26px no-repeat;cursor:pointer}div.light_square .pp_contract:hover{background:url(../images/light_square/sprite.png) 0 -47px no-repeat;cursor:pointer}div.light_square .pp_close{background:url(../images/light_square/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:75px}div.light_square .pp_nav .pp_play{background:url(../images/light_square/sprite.png) -1px -100px no-repeat;height:15px;width:14px}div.light_square .pp_nav .pp_pause{background:url(../images/light_square/sprite.png) -24px -100px no-repeat;height:15px;width:14px}div.light_square .pp_arrow_previous{background:url(../images/light_square/sprite.png) 0 -71px no-repeat}div.light_square .pp_arrow_next{background:url(../images/light_square/sprite.png) -22px -71px no-repeat}div.light_square .pp_next:hover{background:url(../images/light_square/btnNext.png) center right no-repeat;cursor:pointer}div.light_square .pp_previous:hover{background:url(../images/light_square/btnPrevious.png) center left no-repeat;cursor:pointer}div.facebook .pp_top .pp_left{background:url(../images/facebook/sprite.png) -88px -53px no-repeat}div.facebook .pp_top .pp_middle{background:url(../images/facebook/contentPatternTop.png) top left repeat-x}div.facebook .pp_top .pp_right{background:url(../images/facebook/sprite.png) -110px -53px no-repeat}div.facebook .pp_content_container .pp_left{background:url(../images/facebook/contentPatternLeft.png) top left repeat-y}div.facebook .pp_content_container .pp_right{background:url(../images/facebook/contentPatternRight.png) top right repeat-y}div.facebook .pp_expand{background:url(../images/facebook/sprite.png) -31px -26px no-repeat;cursor:pointer}div.facebook .pp_expand:hover{background:url(../images/facebook/sprite.png) -31px -47px no-repeat;cursor:pointer}div.facebook .pp_contract{background:url(../images/facebook/sprite.png) 0 -26px no-repeat;cursor:pointer}div.facebook .pp_contract:hover{background:url(../images/facebook/sprite.png) 0 -47px no-repeat;cursor:pointer}div.facebook .pp_close{background:url(../images/facebook/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:22px}div.facebook .pp_description{margin:0 37px 0 0}div.facebook .pp_loaderIcon{background:url(../images/facebook/loader.gif) center center no-repeat}div.facebook .pp_arrow_previous{background:url(../images/facebook/sprite.png) 0 -71px no-repeat;height:22px;margin-top:0;width:22px}div.facebook .pp_arrow_previous.disabled{background-position:0 -96px;cursor:default}div.facebook .pp_arrow_next{background:url(../images/facebook/sprite.png) -32px -71px no-repeat;height:22px;margin-top:0;width:22px}div.facebook .pp_arrow_next.disabled{background-position:-32px -96px;cursor:default}div.facebook .pp_nav{margin-top:0}div.facebook .pp_nav p{font-size:15px;padding:0 3px 0 4px}div.facebook .pp_nav .pp_play{background:url(../images/facebook/sprite.png) -1px -123px no-repeat;height:22px;width:22px}div.facebook .pp_nav .pp_pause{background:url(../images/facebook/sprite.png) -32px -123px no-repeat;height:22px;width:22px}div.facebook .pp_next:hover{background:url(../images/facebook/btnNext.png) center right no-repeat;cursor:pointer}div.facebook .pp_previous:hover{background:url(../images/facebook/btnPrevious.png) center left no-repeat;cursor:pointer}div.facebook .pp_bottom .pp_left{background:url(../images/facebook/sprite.png) -88px -80px no-repeat}div.facebook .pp_bottom .pp_middle{background:url(../images/facebook/contentPatternBottom.png) top left repeat-x}div.facebook .pp_bottom .pp_right{background:url(../images/facebook/sprite.png) -110px -80px no-repeat}div.pp_pic_holder a:focus{outline:0}div.pp_overlay{background:#000;display:none;left:0;position:absolute;top:0;width:100%;z-index:9500}div.pp_pic_holder{display:none;position:absolute;width:100px;z-index:10000}.pp_content{height:40px;min-width:40px}* html .pp_content{width:40px}.pp_content_container{position:relative;text-align:left;width:100%}.pp_content_container .pp_left{padding-left:20px}.pp_content_container .pp_right{padding-right:20px}.pp_content_container .pp_details{float:left;margin:10px 0 2px}.pp_description{display:none;margin:0}.pp_social{float:left;margin:0}.pp_social .facebook{float:left;margin-left:5px;overflow:hidden;width:55px}.pp_social .twitter{float:left}.pp_nav{clear:right;float:left;margin:3px 10px 0 0}.pp_nav p{float:left;margin:2px 4px;white-space:nowrap}.pp_nav .pp_play,.pp_nav .pp_pause{float:left;margin-right:4px;text-indent:-10000px}a.pp_arrow_previous,a.pp_arrow_next{display:block;float:left;height:15px;margin-top:3px;overflow:hidden;text-indent:-10000px;width:14px}.pp_hoverContainer{position:absolute;top:0;width:100%;z-index:2000}.pp_gallery{display:none;left:50%;margin-top:-50px;position:absolute;z-index:10000}.pp_gallery div{float:left;overflow:hidden;position:relative}.pp_gallery ul{float:left;height:35px;margin:0 0 0 5px;padding:0;position:relative;white-space:nowrap}.pp_gallery ul a{border:1px rgba(0,0,0,.5) solid;display:block;float:left;height:33px;overflow:hidden}.pp_gallery ul a img{border:0}.pp_gallery li{display:block;float:left;margin:0 5px 0 0;padding:0}.pp_gallery li.default a{background:url(../images/facebook/default_thumbnail.gif) 0 0 no-repeat;display:block;height:33px;width:50px}.pp_gallery .pp_arrow_previous,.pp_gallery .pp_arrow_next{margin-top:7px!important}a.pp_next{background:url(../images/light_rounded/btnNext.png) 10000px 10000px no-repeat;display:block;float:right;height:100%;text-indent:-10000px;width:49%}a.pp_previous{background:url(../images/light_rounded/btnNext.png) 10000px 10000px no-repeat;display:block;float:left;height:100%;text-indent:-10000px;width:49%}a.pp_expand,a.pp_contract{cursor:pointer;display:none;height:20px;position:absolute;right:30px;text-indent:-10000px;top:10px;width:20px;z-index:20000}a.pp_close{display:block;line-height:22px;position:absolute;right:0;text-indent:-10000px;top:0}.pp_loaderIcon{display:block;height:24px;left:50%;margin:-12px 0 0 -12px;position:absolute;top:50%;width:24px}#pp_full_res{line-height:1!important}#pp_full_res .pp_inline{text-align:left}#pp_full_res .pp_inline p{margin:0 0 15px}div.ppt{color:#fff;display:none;font-size:17px;margin:0 0 5px 15px;z-index:9999}div.pp_default .pp_content,div.light_rounded .pp_content{background-color:#fff}div.pp_default #pp_full_res .pp_inline,div.light_rounded .pp_content .ppt,div.light_rounded #pp_full_res .pp_inline,div.light_square .pp_content .ppt,div.light_square #pp_full_res .pp_inline,div.facebook .pp_content .ppt,div.facebook #pp_full_res .pp_inline{color:#000}div.pp_default .pp_gallery ul li a:hover,div.pp_default .pp_gallery ul li.selected a,.pp_gallery ul a:hover,.pp_gallery li.selected a{border-color:#fff}div.pp_default .pp_details,div.light_rounded .pp_details,div.dark_rounded .pp_details,div.dark_square .pp_details,div.light_square .pp_details,div.facebook .pp_details{position:relative}div.light_rounded .pp_top .pp_middle,div.light_rounded .pp_content_container .pp_left,div.light_rounded .pp_content_container .pp_right,div.light_rounded .pp_bottom .pp_middle,div.light_square .pp_left,div.light_square .pp_middle,div.light_square .pp_right,div.light_square .pp_content,div.facebook .pp_content{background:#fff}div.light_rounded .pp_description,div.light_square .pp_description{margin-right:85px}div.light_rounded .pp_gallery a.pp_arrow_previous,div.light_rounded .pp_gallery a.pp_arrow_next,div.dark_rounded .pp_gallery a.pp_arrow_previous,div.dark_rounded .pp_gallery a.pp_arrow_next,div.dark_square .pp_gallery a.pp_arrow_previous,div.dark_square .pp_gallery a.pp_arrow_next,div.light_square .pp_gallery a.pp_arrow_previous,div.light_square .pp_gallery a.pp_arrow_next{margin-top:12px!important}div.light_rounded .pp_arrow_previous.disabled,div.dark_rounded .pp_arrow_previous.disabled,div.dark_square .pp_arrow_previous.disabled,div.light_square .pp_arrow_previous.disabled{background-position:0 -87px;cursor:default}div.light_rounded .pp_arrow_next.disabled,div.dark_rounded .pp_arrow_next.disabled,div.dark_square .pp_arrow_next.disabled,div.light_square .pp_arrow_next.disabled{background-position:-22px -87px;cursor:default}div.light_rounded .pp_loaderIcon,div.light_square .pp_loaderIcon{background:url(../images/light_rounded/loader.gif) center center no-repeat}div.dark_rounded .pp_top .pp_middle,div.dark_rounded .pp_content,div.dark_rounded .pp_bottom .pp_middle{background:url(../images/dark_rounded/contentPattern.png) top left repeat}div.dark_rounded .currentTextHolder,div.dark_square .currentTextHolder{color:#c4c4c4}div.dark_rounded #pp_full_res .pp_inline,div.dark_square #pp_full_res .pp_inline{color:#fff}.pp_top,.pp_bottom{height:20px;position:relative}* html .pp_top,* html .pp_bottom{padding:0 20px}.pp_top .pp_left,.pp_bottom .pp_left{height:20px;left:0;position:absolute;width:20px}.pp_top .pp_middle,.pp_bottom .pp_middle{height:20px;left:20px;position:absolute;right:20px}* html .pp_top .pp_middle,* html .pp_bottom .pp_middle{left:0;position:static}.pp_top .pp_right,.pp_bottom .pp_right{height:20px;left:auto;position:absolute;right:0;top:0;width:20px}.pp_fade,.pp_gallery li.default a img{display:none}<br />
<br />
</style><br />
<script type="text/javascript"><br />
<br />
/*<br />
* Superfish v1.4.8 - jQuery menu widget<br />
* Copyright (c) 2008 Joel Birch<br />
*<br />
* Dual licensed under the MIT and GPL licenses:<br />
* http://www.opensource.org/licenses/mit-license.php<br />
* http://www.gnu.org/licenses/gpl.html<br />
*<br />
* CHANGELOG: http://users.tpg.com.au/j_birch/plugins/superfish/changelog.txt<br />
*/<br />
<br />
;(function($){<br />
$.fn.superfish = function(op){<br />
<br />
var sf = $.fn.superfish,<br />
c = sf.c,<br />
$arrow = $(['<span class="',c.arrowClass,'"> &#187;</span>'].join('')),<br />
over = function(){<br />
var $$ = $(this), menu = getMenu($$);<br />
clearTimeout(menu.sfTimer);<br />
$$.showSuperfishUl().siblings().hideSuperfishUl();<br />
},<br />
out = function(){<br />
var $$ = $(this), menu = getMenu($$), o = sf.op;<br />
clearTimeout(menu.sfTimer);<br />
menu.sfTimer=setTimeout(function(){<br />
o.retainPath=($.inArray($$[0],o.$path)>-1);<br />
$$.hideSuperfishUl();<br />
//if (o.$path.length &amp;&amp; $$.parents(['li.',o.hoverClass].join('')).length<1){over.call(o.$path);}<br />
if (o.$path.length) {<br />
if ($$.parents(['li.',o.hoverClass].join('')).length<1){over.call(o.$path);}<br />
}<br />
},o.delay); <br />
},<br />
getMenu = function($menu){<br />
var menu = $menu.parents(['ul.',c.menuClass,':first'].join(''))[0];<br />
sf.op = sf.o[menu.serial];<br />
return menu;<br />
},<br />
addArrow = function($a){ $a.addClass(c.anchorClass).append($arrow.clone()); };<br />
<br />
return this.each(function() {<br />
var s = this.serial = sf.o.length;<br />
var o = $.extend({},sf.defaults,op);<br />
o.$path = $('li.'+o.pathClass,this).slice(0,o.pathLevels).each(function(){<br />
$(this).addClass([o.hoverClass,c.bcClass].join(' '))<br />
.filter('li:has(ul)').removeClass(o.pathClass);<br />
});<br />
sf.o[s] = sf.op = o;<br />
var bcheck = false;<br />
if ($.fn.hoverIntent) {<br />
if (!o.disableHI) bcheck = true;<br />
}<br />
<br />
$('li:has(ul)',this)[(bcheck) ? 'hoverIntent' : 'hover'](over,out).each(function() {<br />
if (o.autoArrows) addArrow( $('>a:first-child',this) );<br />
})<br />
.not('.'+c.bcClass)<br />
.hideSuperfishUl();<br />
<br />
var $a = $('a',this);<br />
$a.each(function(i){<br />
var $li = $a.eq(i).parents('li');<br />
$a.eq(i).focus(function(){over.call($li);}).blur(function(){out.call($li);});<br />
});<br />
o.onInit.call(this);<br />
<br />
}).each(function() {<br />
var menuClasses = [c.menuClass];<br />
//if (sf.op.dropShadows &amp;&amp; !($.browser.msie &amp;&amp; $.browser.version < 7)) menuClasses.push(c.shadowClass);<br />
if (sf.op.dropShadows) if (!$.browser.msie) if (!($.browser.version < 7)) menuClasses.push(c.shadowClass);<br />
$(this).addClass(menuClasses.join(' '));<br />
});<br />
};<br />
<br />
var sf = $.fn.superfish;<br />
sf.o = [];<br />
sf.op = {};<br />
sf.IE7fix = function(){<br />
var o = sf.op;<br />
//if ($.browser.msie &amp;&amp; $.browser.version > 6 &amp;&amp; o.dropShadows &amp;&amp; o.animation.opacity!=undefined)<br />
if ($.browser.msie) if($.browser.version > 6) if (o.dropShadows) if (o.animation.opacity!=undefined)<br />
this.toggleClass(sf.c.shadowClass+'-off');<br />
};<br />
sf.c = {<br />
bcClass : 'sf-breadcrumb',<br />
menuClass : 'sf-js-enabled',<br />
anchorClass : 'sf-with-ul',<br />
arrowClass : 'sf-sub-indicator',<br />
shadowClass : 'sf-shadow'<br />
};<br />
sf.defaults = {<br />
hoverClass : 'sfHover',<br />
pathClass : 'overideThisToUse',<br />
pathLevels : 1,<br />
delay : 800,<br />
animation : {opacity:'show'},<br />
speed : 'normal',<br />
autoArrows : true,<br />
dropShadows : true,<br />
disableHI : false, // true disables hoverIntent detection<br />
onInit : function(){}, // callback functions<br />
onBeforeShow: function(){},<br />
onShow : function(){},<br />
onHide : function(){}<br />
};<br />
$.fn.extend({<br />
hideSuperfishUl : function(){<br />
var o = sf.op,<br />
not = (o.retainPath===true) ? o.$path : '';<br />
o.retainPath = false;<br />
var $ul = $(['li.',o.hoverClass].join(''),this).add(this).not(not).removeClass(o.hoverClass)<br />
.find('>ul').hide().css('visibility','hidden');<br />
o.onHide.call($ul);<br />
return this;<br />
},<br />
showSuperfishUl : function(){<br />
var o = sf.op,<br />
sh = sf.c.shadowClass+'-off',<br />
$ul = this.addClass(o.hoverClass)<br />
.find('>ul:hidden').css('visibility','visible');<br />
sf.IE7fix.call($ul);<br />
o.onBeforeShow.call($ul);<br />
$ul.animate(o.animation,o.speed,function(){ sf.IE7fix.call($ul); o.onShow.call($ul); });<br />
return this;<br />
}<br />
});<br />
<br />
})(jQuery);<br />
<br />
/*<br />
* Supersubs v0.2b - jQuery plugin<br />
* Copyright (c) 2008 Joel Birch<br />
*<br />
* Dual licensed under the MIT and GPL licenses:<br />
* http://www.opensource.org/licenses/mit-license.php<br />
* http://www.gnu.org/licenses/gpl.html<br />
*<br />
*<br />
* This plugin automatically adjusts submenu widths of suckerfish-style menus to that of<br />
* their longest list item children. If you use this, please expect bugs and report them<br />
* to the jQuery Google Group with the word 'Superfish' in the subject line.<br />
*<br />
*/<br />
<br />
(function($){ // $ will refer to jQuery within this closure<br />
<br />
$.fn.supersubs = function(options){<br />
var opts = $.extend({}, $.fn.supersubs.defaults, options);<br />
// return original object to support chaining<br />
return this.each(function() {<br />
// cache selections<br />
var $$ = $(this);<br />
// support metadata<br />
var o = $.meta ? $.extend({}, opts, $$.data()) : opts;<br />
// get the font size of menu.<br />
// .css('fontSize') returns various results cross-browser, so measure an em dash instead<br />
var fontsize = $('<li id="menu-fontsize">&#8212;</li>').css({<br />
'padding' : 0,<br />
'position' : 'absolute',<br />
'top' : '-999em',<br />
'width' : 'auto'<br />
}).appendTo($$).width(); //clientWidth is faster, but was incorrect here<br />
// remove em dash<br />
$('#menu-fontsize').remove();<br />
// cache all ul elements<br />
$ULs = $$.find('ul');<br />
// loop through each ul in menu<br />
$ULs.each(function(i) { <br />
// cache this ul<br />
var $ul = $ULs.eq(i);<br />
// get all (li) children of this ul<br />
var $LIs = $ul.children();<br />
// get all anchor grand-children<br />
var $As = $LIs.children('a');<br />
// force content to one line and save current float property<br />
var liFloat = $LIs.css('white-space','nowrap').css('float');<br />
// remove width restrictions and floats so elements remain vertically stacked<br />
var emWidth = $ul.add($LIs).add($As).css({<br />
'float' : 'none',<br />
'width' : 'auto'<br />
})<br />
// this ul will now be shrink-wrapped to longest li due to position:absolute<br />
// so save its width as ems. Clientwidth is 2 times faster than .width() - thanks Dan Switzer<br />
.end().end()[0].clientWidth / fontsize;<br />
// add more width to ensure lines don't turn over at certain sizes in various browsers<br />
emWidth += o.extraWidth;<br />
// restrict to at least minWidth and at most maxWidth<br />
if (emWidth > o.maxWidth) { emWidth = o.maxWidth; }<br />
else if (emWidth < o.minWidth) { emWidth = o.minWidth; }<br />
emWidth += 'em';<br />
// set ul to width in ems<br />
$ul.css('width',emWidth);<br />
// restore li floats to avoid IE bugs<br />
// set li width to full width of this ul<br />
// revert white-space to normal<br />
$LIs.css({<br />
'float' : liFloat,<br />
'width' : '100%',<br />
'white-space' : 'normal'<br />
})<br />
// update offset position of descendant ul to reflect new width of parent<br />
.each(function(){<br />
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<article><br />
<h1><p>Lab Protocol</p></h1><br />
</font><br />
<font face="Arial, Helvetica"><br />
<p><a href="#Site-Directed_Mutagenesis">1. Site-Directed Mutagenesis</a></p><br />
<p><a href="#Restriction">2. Mutation Verification by Restriction Enzyme Digestion</a></p><br />
<p><a href="#Media">3. Media Preparation</a></p><br />
<p><a href="#Bacterial">4. Bacterial Transformation</a></p><br />
<p><a href="#Colony">5. Colony PCR for Verification</a></p><br />
<p><a href="#Culture">6. Cultivate the Bacteria</a></p><br />
<p><a href="#Plasmid">7. Plasmid DNA Isolation</a></p><br />
<p><a href="#Restriction_2">8. Mutation Verification by Restriction Enzyme Digestion </a></p><br />
<p><a href="#Amplify">9. Polymerase Chain Reaction and Electrophoresis</a></p><br />
<p><a href="#Electrophoresis">10. Double Restriction Enzyme Digestion and Electrophoresis </a></p><br />
<p><a href="#Ligation">11. Ligation</a></p><br />
<p><a href="#Bacterial_Transformation">12. Bacterial Transformation</a></p><br />
<p><a href="#bacterial_colony">13. Bacterial Colony PCR</a></p><br />
<p><a href="#Culture_the">14. Cultivate the Bacteria</a></p><br />
<p><a href="#Plasmid_DNA">15. Plasmid DNA Isolation</a></p><br />
<p><a href="#Restriction_Enzyme">16. Restriction Enzyme Digestion and Electrophoresis</a></p><br />
<p><a href="#Ligation1">17. Ligation</a></p><br />
<p><a href="#Bacteria">18. Bacteria Transformation</a></p><br />
<p><a href="#Cultivate">19. Cultivate the Bacteria</a></p><br />
<p><a href="#Flow">20. Flow Cytometer Analysis</a></p><br />
<p><a href="#Fluorescence">21. Fluorescence Microscope </a></p><br />
<br><br />
<a name="Site-Directed_Mutagenesis" ></a></font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Site-Directed Mutagenesis</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Plasmid pSB1A3 was chosen as a backbone vector for cloning. The Pst I site in pSB1A3 was mutated to Afl II site to facilitate following cloning processes. Proper primers were designed and PCR-based site-directed mutageneis were carried out to generate this mutation as descbibed below. </p><br />
<br />
<p><strong>Method:</strong></p><br />
<br />
<p>1. Set up PCR assay tubes as described below:<br />
<br/><br />
Total: 25 μl<br /><br />
+ 0.25 μl of Ex Taq polymerase <br /><br />
+ 2.5 μl of 10× Taq reaction buffer<br /><br />
+ 2.0 μl of dNTP(2 mM) <br /><br />
+ 1.0 μl of template (E.coli plasmid 817) <br /><br />
+ 1.0 μl of oligonucleotide primer PtoA-F*<br /><br />
+ 1.0 μl of oligonucleotide primer PtoA-R* <br /><br />
+ 18.25 μl of ddH2O <br /><br />
*The sequences of primer pair PtoA-F and PtoA-R.<br /><br />
PtoA-F 5'-CCACCTGACGTCTAAGAAAC-3'<br /><br />
PtoA-R 5'-ATGATCATCGCCGGCGAATTCAGGC-3' <br /><br />
</p><br />
<p> 2. Set parameters for PCR to amplify desired products. </p><br />
<br />
<table><br />
<tr><br />
<td>Temperature</td> <td>Time</td> <td>Cycle</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>13.5min</td> <td>1</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>55˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>72˚C</td> <td>1min 20sec</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>4˚C</td> <td>7hrs</td> <td>1</td><br />
</tr><br />
</table><br />
<br />
<font face="Arial, Helvetica"><br />
<p><br><br />
<a name="Restriction" ></a> </p><br />
</font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Restriction Enzyme Digestion for Verification</font></b></font></h3><br />
<p align="left">To verify whether PstI site in pSB1A3 was successfully mutated to AflII site, we performed restriction enzyme digestion experiments. </p><br />
<p>Bacterial plasmids are double-stranded circular DNA molecules and uncut plasmid DNA can be in any of three forms - nicked circular, linear, closed supercoiled. When run on an agarose gel one frequently will see these forms as different bands with closed supercoiled form migrates the fastest, linear form migrates the slowest, and nicked circular migrates in between. If the PStI site was successfully mutated to AflII site, we expected to see increased linear form of pSB1A3 when digested with AflII. SpeI-cut pSB1A3 was served as a positive control. </p><br /><br />
<strong>Method</strong><br /><br />
1. Set up Pst I digestion in 1.5 ml eppendorf tubes as described below:<br /><br />
Total: 10μl<br /><br />
+ 0.5μl of Pst I restriction enzyme (company :Takara)<br /><br />
+ 1μl of 10XH buffer<br /><br />
+ 1μl of plasmid DNA<br /><br />
+ 7.5μl of ddH2O <br /><br />
2. Set up Afl II digestion in 1.5 ml eppendorf tubes as described below:<br /><br />
Total: 10μl<br /><br />
+ 0.5μl of Afl II restriction enzyme, (company :Takara)<br /><br />
+ 1μl of 10XM buffer<br /><br />
+ 1μl of plasmid DNA<br /><br />
+ 1.0μl of 0.01% BSA<br /><br />
+ 6.5μl of ddH2O<br /><br />
3. Incubate the eppendorf tubes in 37℃ water bath for 1-2 hr. <br/><br />
4. Prepare 1% agarose gel in Conical flask. Weigh 0.6 g agarose and add 60 ml 1x TAE (diluted from 50x TAE). Cover the Conical flask with silver paper to avoid the loss of water vapor. Place the Conical flask in the microwave and microwave for 1 minute with a middle power. Take it out and shake gently till the solution is homogeneous,(BE CAREFUL to watch the solution closely when shake it–it superheats and can boil over and cause severe burns). Continue microwave and swirl until solution is seen clear and homogeneous with no existence of solid. After cool down the agarose gel briefly, add 3 μl of Gelred (10000x ) and mix well. Pour the agarose gel in gel casting apparatus and insert combs.<br/><br />
5. By inserting the pipette tip below the TAE liquid and into the well, add 5 μl of 1kb DNA ladder solution to first (and last if desired) well, skip one well, then begin adding the 5μl of digested DNA solutions mixed with 1 μl loading buffer (6x) to the wells. <br/><br />
6. Place the cover on the electrophoresis unit, plug into the power source, and turn on voltage to 120V, set time to 30 minutes, and press the start button twice,until the bubbles are seen. DNA separation can be observed as time goes on by turning off the power supply then gently removing the basin from the electrophoresis unit (be careful not to let the gel slip out of the basin) and placing on the UV transilluminator to see DNA bands. <br/><br />
7. When the desired level of separation is obtained, the basin can be placed on the transilluminator for picture taking(Of the absence of transilluminator,we use camera to take pictures with the UV light ). <br/><br />
8. Cut the gel of specific position and collect it in tubes that have measured weight. <br/><br />
9. Use DNA Gel Extraction Ki to purify the plasmid DNA mutant-psb1a3.<br />
RPF (SpeI/AflII-digested), GFP (SpeI/AflII-digested) fragments were ligated into this vector, which was followed by insertion of designed terminator sequences between RFP and GFP, respectively.</p><br />
<font face="Arial, Helvetica"><br />
<p> <img src="https://static.igem.org/mediawiki/2012/5/59/11.png" alt="" class="img_fl img_border" align="left"/> </p><br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<p>(Figure 1 : This figure shows that the site-directed mutagenesis succeed ,we successfully change a restriction enzyme cutting site named Pst I to Afl II. Lane 1 represents the plasmid mutant-psb1a3, lane 2 shows that the mutant-psb1a3 cannot be digested by restriction enzyme Spe I ,lane 3 shows that mutant-psb1a3 can be digested by restriction enzyme Afl II.) </p><br />
<br />
<br />
<a name="Media"></a></font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Media Preparation</font></b></font></h3><br />
<p align="left">For all experiments involving the bacterial biomass and experimentation, proper media is chosen to grow the cells. Commonly,we use Lysogeny broth media for <em>E. coli</em>. The following is the media compositions and their quantities.<br /><br />
<p><strong>Method:</strong></p><br />
<p><br />
1.Prepare the Lysogeny Broth (LB) liquid media (1 L) as indicated below: <br/><br />
+ Bacto-Tryptone - 10 g<br/><br />
+ NaCl - 10 g<br/><br />
+ Yeast Extract - 5 g<br/><br />
Add ddH2O and 5mmol/L Tris Buffer in a measuring cylinder to ensure accuracy, to make a total of 1 liter and pH is 8.0.<br/><br />
2.Prepare the Lysogeny Broth (LB) solid media (1 L) as indicated below:<br/><br />
+ Bacto-Tryptone - 10 g<br/><br />
+ NaCl - 10 g<br/><br />
+ Yeast Extract - 5 g<br/><br />
+ Difco Agar - 15g<br/><br />
Add ddH2O and 5mmol/L Tris Buffer in a measuring cylinder to ensure accuracy, to make a total of 1 liter and pH is 8.0.<br/><br />
3. Autoclaving<br/><br />
Autoclave at 121 °C for 60 minutes. After the media cooling down enough, antibiotics Ampicillin(100mg of Ampicillin per 1ml of the media) are added. At last the media are poured 15ml on each plate and become solid.Store the plate at 4℃ refrigerator.<br />
</p><br />
<br />
<font face="Arial, Helvetica"><br />
<p>&nbsp;</p><br />
<br><br />
<a name="Bacterial"></a></font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Bacterial Transformation</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Transformation is commonly used to introduce recombinant plasmid DNA into bacterial strains which can transform naturally or can be made competitive for transformation by artificial means. The purpose of this technique is to introduce a recombinant plasmid DNA into a bacterial strains and to use bacteria strains to amplify the plasmid mutant-pSB1A3 for further plasmid construction.</p><br />
<br />
<strong>Method</strong><br /><br />
1. Take out an appropriate number of tubes that contain competent cells(100μl ) from the freezer. Immediately place the tubes on ice, so that all but the cap is surrounded by ice. Allow the cells to thaw on ice for 2-5 min.<br /><br />
2. Visually check the cells to see whether they have thawed and gently flick the cells 1-2 times to evenly resuspend the cells.<br /><br />
3. Add 10μl PCR products(mini-prep purified) to the competent cells DH-5α. Stir gently to mix and return the tube to the ice, making sure that the tube is surrounded by ice except for the cap. Repeat for additional two times for the same samples.<br /><br />
4. Incubate the tubes on ice for 30 min.<br /><br />
5. Place the tubes in a 42°C water bath for exactly 90 sec; do not shake.<br /><br />
6. Place the tubes on ice for 2 min to cool down.<br /><br />
7. Add 800 l of room temperature LB medium to each tube.<br /><br />
8. Shake the tubes vigorously at 37<a name="OLE_LINK2" id="OLE_LINK2"></a><a name="OLE_LINK1" id="OLE_LINK1">°C</a> for 45-60 min.<br /><br />
9. Centrifuge the tubes at 3K RPM for 1 min. Discard the supernatant liquor and leave 100-200 μl of the mixtures.Mix the contents and spread the whole liquid on LB agar plates containing the appropriate antibiotic ampicillin for the plasmid.<br /><br />
10. Place the plates on the bench for several min to allow excess liquid to be absorbed, and then invert and incubate overnight at 37°C (12-16 h).</p><br />
<br><br />
<a name="Colony"></a></font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Colony PCR for Verification</font> </b></font></h3><br />
<font face="Arial, Helvetica"><br />
<P>Colony PCR is used to identify and select cell colonies that have the correct plasmid inserted. The procedure is a way to do several PCR operations on cell colonies in parallel, to evaluate the results and select the corresponding positive cell colonies. After an overnight growth of E.coli, we can pick up several colonies from the plate and do a colony PCR verification. Besides, the colonies we choose and should also be stored, we can incubate these colonies in one plate after every colony has been marked.</p><br />
<br />
<strong>Method</strong><br /><br />
1. Prepare the sample reaction as indicated below:<br /><br />
Total: 25μl <br /><br />
+ 0.25 μl of Ex Taq polymerase (company:Takara)<br /><br />
+ 2.5 μl of 10× Taq reaction buffer<br /><br />
+ 1.0 μl of R-NPS-F*<br /><br />
+ 1.0 μl of G-SXA-R*<br /><br />
+ 1.0 μl of plasmid mutant-psb1a3<br /><br />
+ 2.0 μl of dNTP( 25mM ) <br /><br />
+ 17.25 μl of ddH2O <br /><br />
Note: The sequences of primers R-NPS-F , G-SXA-R <br /><br />
R-NPS-F 5'-TATAGCGGCCGCCTTAAGTAAGTAAGAGTATACG-3' <br /><br />
G-SXA-R 5'-TCTCCGACTTAAGGGATCCTATAAACGCAG-3'<br /><br />
<br />
<p> 2. Set parameters for PCR to amplify desired products. </p><br />
<br />
<table><br />
<tr><br />
<td>Temperature</td> <td>Time</td> <td>Cycle</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>13.5min</td> <td>1</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>55˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>72˚C</td> <td>1min 20sec</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>4˚C</td> <td>7hrs</td> <td>1</td><br />
</tr><br />
</table><br />
<br />
3. Electrophorese the total system and observe the lane separation. </P><br />
<br><br />
<a name="Culture"></a></font><br />
<br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Cultivate the Bacteria</b></font></h3><br />
<font face="Arial, Helvetica"><p>According to the results of the PCR detection, positive colonies are chosen and transferred them to 5ml LB liquid media ( 5μl of ampicillin added) stored in 12.5ml centrifuge tubes. Put the centrifuge tubes in 37℃ gas bath overnight. </p><br />
<br><br />
<a name="Plasmid"></a><br />
<h3><b>Plasmid DNA Isolation</b></h3><br />
<p>Use E.Z.N.A.TM Plasmid Mini I to realize plasmid DNA isolation.<br /><br />
<strong>Method</strong></p><br />
<ul><br />
<ul><br />
<li>Transfer 5 ml of overnight culture into a 1.5-ml eppendorf tube labeled with group number. </li><br />
<li>Centrifuge the sample at max. speed of desk top centrifuge and RT for 1min to pellet the cells.</li><br />
<li>Discard the supernatant. Remove as much of the supernatant as possible without disturbing the cell pellet. </li><br />
<li>Repeat step 1 and 2 twice. </li><br />
<li>Resuspend the pellet completely in 250 ml of Solution I (containing RNase A) by vortexing the samples vigorously . No clumps should be visible in the tube. </li><br />
<li>Add 250 ml of Solution II and mix the sample by gently inverting the tube 4 to 6 times. Do not vortex or shake the sample vigorously. The bacterial suspension should begin to clear which have lysed the bacterial cells in this step. <strong>Warning: </strong>Do not stop here for more than five min, as the high pH hurts your DNA! </li><br />
<li>Add 350 ml of Solution III and mix by gently inverting the tube 4 to 6 times until a flocculent white precipitate forms. Do not shake vigorously, as it might break the genomic DNA. </li><br />
<li>Centrifuge at maximum speed for 10 min at room temperature to pellet the cell debris. You should see a white precipitate in the tube after the centrifugation. </li><br />
<li>While the samples are centrifuging, for each sample, label a clean HiBind Miniprep Column which is to assembled in a 2-ml collection tube</li><br />
<li>Apply the supernatants from step 8 to the columns. </li><br />
<li>Centrifuge at maximum speed for 1 min at RT. Discard the flow-through in the collection tube. </li><br />
<li>Add 500 ml of Buffer HB to wash the Hibind Miniprep Column. Centrifuge at maximum speed for 1 min at RT. Discard the flow-through in the collection tube. </li><br />
<li>Wash the column by adding 700 ml of DNA Wash Buffer diluted with absolute ethanol. Centrifuge at maximum speed for 1 min at room temperature and discard the flow-through.</li><br />
<li>Then centrifuge the tubes again for 2 min to remove all the moisture.</li><br />
<li>Place the column in a clean 1.5 ml eppendorf tube that is labeled with the plasmid name and group number. To elute the DNA, add 50 ml of Elution Buffer to the center of each column. Let the samples stand for 2 or more minutes at RT, and then centrifuge for 1 min. The sample in the centrifuge tube (bottom) is your plasmid DNA. </li><br />
<li>Discard the column and save the sample in the eppendorf tube by placing it in the freezer (-20°C). </li><br />
</ul></ul><br />
<a name="#Restriction_2" ></a> </font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Restriction Enzyme Digestion and Electrophoresis</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Because the colony PCR test is so sensitive and affect markedly by environment factors. So we do a restriction enzyme digestion to ensure that the isolated plasmid is the site-directed mutated plasmid.<br /><br />
<strong>Method</strong><br /><br />
1. Prepare the control reaction as indicated below:<br /><br />
Total: 10μl<br /><br />
+ 0.5μl of Pst I restriction enzyme(company :Takara)<br /><br />
+ 1μl of 10XH buffer<br /><br />
+ 1μl of plasmid DNA<br /><br />
+ 7.5μl of ddH2O <br /><br />
2. Prepare the sample reaction as indicated below:<br /><br />
Total: 10μl<br /><br />
+ 0.5μl of Afl II restriction enzyme, (company :Takara)<br /><br />
+ 1μl of 10XM buffer<br /><br />
+ 1μl of plasmid DNA<br /><br />
+ 1.0μl of 0.01% BSA<br /><br />
+ 6.5μl of ddH2O <br /><br />
3. Electrophorese the total system and observe the lane separation.</p><br />
<br><br />
<a name="Amplify"></a> </font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Polymerase Chain Reaction(GFP &amp; RFP) and Electrophoresis</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>GFP and RFP DNA fragments are the insert which need to be ligate to the plasmid mutant-psb1a3. Do a PCR amplification can get enough quantities for the following reactions.<br /><br />
<strong>Method </strong><br /><br />
1.Prepare the sample reaction as indicated below:<br /><br />
Total: 100μl ( PCR <a name="OLE_LINK37" id="OLE_LINK37"></a><a name="OLE_LINK36" id="OLE_LINK36">amplification</a> of GFP fragments) <br /><br />
+ 1.0μl of Taq DNA polymerase,#EP0402<br /><br />
+ 10μl of 10XTaq buffer <br /><br />
+ 10μl of MgCl2(25mM)<br /><br />
+ 10μl of dNTP(2mM)<br /><br />
+ 2μl of G-SXA-R* <br /><br />
+ 2μl of G-SXA-F*<br /><br />
+ 4μl of DNA template<br /><br />
+ 61μl of ddH2O <br /><br />
Total: 100μl(PCR amplification of RFP fragments) <br /><br />
+ 1.0μl of Taq DNA polymerase, EP0402<br /><br />
+ 10μl of 10XTaq buffer <br /><br />
+ 10μl of MgCl2(25mM)<br /><br />
+ 10μl of dNTP(2mM)<br /><br />
+ 2μl of R-NPS-R* <br /><br />
+ 2μl of R-NPS-F*<br /><br />
+ 4μl of DNA template<br /><br />
+ 61μl of ddH2O <br /><br />
<br />
Note: The sequences of primers R-NPS-F , R-NPS-R , G-SXA-F ,G-SXA-R <br /><br />
R-NPS-F 5'-TATAGCGGCCGCCTTAAGTAAGTAAGAGTATACG-3' <br /><br />
R-NPS-R 5'-CGGAGACTAGTCTGCAGATCACATAAGTAAAGTGATAATC-3' <br /><br />
G-SXA-F 5'-CTAGACTAGTTCTAGAGGCGGACTCACTATAGA-3' <br /><br />
G-SXA-R 5'-TCTCCGACTTAAGGGATCCTATAAACGCAG-3'<br /><br />
<br />
<p> 2. Set parameters for PCR to amplify desired products. </p><br />
<br />
<table><br />
<tr><br />
<td>Temperature</td> <td>Time</td> <td>Cycle</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>13.5min</td> <td>1</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>55˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>72˚C</td> <td>1min 20sec</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>4˚C</td> <td>7hrs</td> <td>1</td><br />
</tr><br />
</table><br />
</p><br />
</font><br />
<ul><br />
<li>Use DNA Gel Extraction Kit to purify the GFP and RFP DNA fragments after the Electrophoresis.</li><br />
</ul><br />
<p align="left"><strong>Figure</strong></p><br />
<p><font face="Arial, Helvetica"><img src="https://static.igem.org/mediawiki/2012/7/72/111.png" alt="" class="img_fl img_border" align="left"/> </font></p><br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<p>(Figure 2 : This figure shows that The PCR reaction system can amplify large quantities of GFP and RFP DNA fragments. The digestion based on the GFP and RFP DNA fragments can be done to prepare for the ligation. Lane 1 represents the template E.coli 817 can amplify the GFP and RFP DNA fragments , lane 2 represents the template E.coli 817(355.5) can also amplify the GFP and RFP DNA fragments.)<br/><br/><br />
<font face="Arial, Helvetica"><br/><br />
<a name="Restriction_Enzyme"></a><br />
</font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Double Restriction Enzyme Digestion and Electrophoresis.</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Use specific restriction enzymes to digest plasmid mutant-psb1a3,GFP and RFP to get sticky ends and purify the DNA fragment after the Electrophoresis.<br /><br />
<strong>Method:</strong></p><br />
</font><br />
<ul><br />
<li> Digestion of plasmid mutant-psb1a3 </li><br />
</ul><br />
<p>Prepare the sample reaction as indicated below:<br /><br />
Total: 50μl <br /><br />
+ 3.0μl of Not I restriction enzyme, #ER0591<br /><br />
+ 3.0μl of Afl II restriction enzyme, #ER0831<br /><br />
+ 5.0μl of 10X buffer O <br /><br />
+ 1.0μl of mutant-psb1a3 plasmid <br /><br />
+ 37.0μl of ddH2O<br /><br />
2. Digestion of PCR product GFP<br /><br />
Prepare the sample reaction as indicated below:<br /><br />
Total: 50μl <br /><br />
+ 3.0μl of Not I restriction enzyme, #ER0591<br /><br />
+ 3.0μl of Spe I restriction enzyme, #ER1251 <br /><br />
+ 5μl of 10X buffer Tango<br /><br />
+ 10μl of PCR products GFP<br /><br />
+ 29μl of ddH2O<br /><br />
3. Digestion of PCR product RFP<br /><br />
Prepare the sample reaction as indicated below:<br /><br />
Total: 50μl <br /><br />
+ 3.0μl of Afl II restriction enzyme, #ER0831<br /><br />
+ 3.0μl of Spe I restriction enzyme, #ER1251 <br /><br />
+ 5μl of 10X buffer Tango<br /><br />
+ 10μl of PCR products RFP<br /><br />
+ 29μl of ddH2O<br /><br />
4. Put the tubes in 37℃ environment for 4-8 hours <br /><br />
5. Use DNA Gel Extraction Kit to purify the mutant-psb1a3 fragment, GFP and RFP after digestion and named them by mutant-psb1a3 (NA) ,GFP(NS) and RFP(AS) after the Electrophoresis.</p><br />
<font face="Arial, Helvetica"><br><br />
<a name="Ligayion"></a> </font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Ligation</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Ligation is the process that target DNA gene is inserted into a plasmid. Both the vector and insert are prepared to have the sticky ends. These two kinds of DNA pieces are placed in a reaction tube and the proper DNA ligase, buffer, and cofactors are added for the reaction to take place. When done properly, the ligation will result in a successful combination of the insert and plasmid into one plasmid.Based on the digestion of mutant-psb1a3,GFP and RFP DNA fragments,also ligation can be done. We ligate mutant-psb1a3 vector and sticky GFP and RFP DNA fragments to construct an new plasmid mutant-psb1a3-GR. <br /><br />
1. Prepare the control reaction as indicated below:<br /><br />
Total: 10μl <br /><br />
+ 1.0μl of plasmid mutant-psb1a3 (NA) <br /><br />
+ 3.0μl of GFP(NS) <br /><br />
+ 3.0μl of RFP(AS) <br /><br />
+ 2.0μl of T4 DNA Ligase,#EL0011 <br /><br />
+ 1.0μl of 10XT4 Ligase buffer<br /><br />
Note: GFP(NS) means the product of GFP DNA fragments digested by restriction enzyme Not I and Spe I. <br /><br />
2. Prepare the sample reaction as indicated below:<br /><br />
Total: 10μl <br /><br />
+ 1.0μl of plasmid mutant-psb1a3 (NA) <br /><br />
+ 2.0μl of T4 DNA Ligase, #EL0011 <br /><br />
+ 1.0μl of 10XT4 Ligase buffer<br /><br />
+ 6.0μl of ddH2O<br /><br />
3. Put the tubes in 22℃ water bath, react for 8-12 hours. <br><br />
<a name="Bacterial_Transformation"></a> </font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Bacterial Transformation</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Transform the ligation products into the DH-5α competent cells, put the plate on 37℃ gas bath for 12-16 hours. </p><br />
<br><br />
<a name="Bacterial_Colony"></a> </font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Bacterial Colony PCR</font></b> </font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Colony PCR is used to identify and select cell colonies that have the correct plasmid insert. This procedure is a way to do several PCR operations on cell colonies in parallel, to evaluate the results and select the corresponding good cell colonies. After an overnight growth of E.coli, we can pick up some colonies from the plate and do a colony PCR verification. Besides, the colonies we choose and should also be stored, we can incubate these colonies in one plate after every colony has been marked.<br /><br />
<strong>Method</strong><br /><br />
1. Prepare the sample reaction as indicated below:<br /><br />
Total: 20μl <br /><br />
+ 0.25 μl of Ex Taq polymerase,#EP0402 <br /><br />
+ 2.0 μl of 10× Taq reaction buffer<br /><br />
+ 1.0 μl of R-NPS-F <br /><br />
+ 1.0 μl of G-SXA-R<br /><br />
+ 5.0 μl of bacterial colony<br /><br />
+ 2.0 μl of dNTP( 25mM ) <br /><br />
+ 8.75 μl of ddH2O <br /><br />
Note: Here listed the sequence of primers.<br /><br />
R-NPS-F 5'-TATAGCGGCCGCCTTAAGTAAGTAAGAGTATACG-3' <br /><br />
R-NPS-R 5'-CGGAGACTAGTCTGCAGATCACATAAGTAAAGTGATAATC-3' <br /><br />
G-SXA-F 5'-CTAGACTAGTTCTAGAGGCGGACTCACTATAGA-3' <br /><br />
G-SXA-R 5'-CCGACTTAAGGGATCCTATAAACGCAG-3'<br /><br />
2. Procedures on the thermocycler are listed below:<br /><br />
① 94˚C for 5 min<br /><br />
② 30 cycle<br /><br />
a. 94˚C for 1 min<br /><br />
b. 55˚C for 1 min<br /><br />
c. 72˚C for 1 min20sec<br /><br />
③ 4℃ for 7 hours </p><br />
</font><br />
<ul><br />
<li> Electrophorese the total system and observe the lane separation. </li><br />
</ul><br />
<p><strong>Figure</strong></p><br />
<p> <img src="https://static.igem.org/mediawiki/2012/0/07/1111.png" alt="" class="img_fl img_border" /> </p><br />
<p>(Figure 3: The lane on the figure are 2k fragments, it shows the GFP and RFP are ligated to the vectors which was isolated from the bacterial colonies.) </p><br />
<p><font face="Arial, Helvetica"><br><br />
<a name="Culture_the"></a><br />
</font></p><br />
<font face="Arial, Helvetica"><br />
</font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Cultivate the Bacteria</b></font></h3><br />
<font face="Arial, Helvetica"><p>According to the results of the PCR detection, we choose positive colonies and transfer them to 5ml LB liquid media ( 5μl of ampicillin has added) stored in 12.5ml centrifuge tubes. Put the centrifuge tubes in 37℃ gas bath overnight. </p><br />
<p><font face="Arial, Helvetica"><a name="Plasmid_DNA"></a> </font></p><br />
<font face="Arial, Helvetica"> </font> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Plasmid DNA Isolation</b></font></h3><br />
<p><font face="Arial, Helvetica">Use E.Z.N.A.TM Plasmid Mini I to isolate the constructed plasmid mutant-psb1a3-GR. </font></p><br />
<p><font face="Arial, Helvetica"><a name="Restriction_Enzyme" ></a> </font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Restriction Enzyme Digestion and Electrophoresis</b></font></h3><br />
<p>From the last step, we got the certain quantities of isolated plasmids. In this step, we do two restriction enzyme digestion reactions, one to prove that the plasmid is construct correctly ( mutant-psb1a3-GR ), one to get sticky ends preparing for the ligation.<br /><br />
<strong>Method</strong></p><br />
<ul><br />
<li><strong>Restriction Enzyme Digestion to prove that plasmid is constructed correctly</strong></li><br />
</ul><br />
<p align="left">a. Prepare the sample reaction as indicated below:<br /><br />
Total: 10μl<br /><br />
+ 1.0μl of Not I restriction enzyme,#ER0591<br /><br />
+ 1.0μl of Spe I restriction enzyme,#ER1251 <br /><br />
+ 2.0μl of Buffer Tango( 10X )<br /><br />
+ 1.5μl of plasmid mutant-psb1a3-GR<br /><br />
+ 14.5μl of ddH2O <br /><br />
b. Electrophorese the total system and observe the lane separation. </p><br />
<ul><br />
<li><strong>Restriction Enzyme Digestion to get sticky ends preparing for the ligation.</strong></li><br />
</ul><br />
<p align="left">a. Prepare the sample reaction as indicated below:<br /><br />
Total: 50μl<br /><br />
+ 5.0μl of Pst I restriction enzyme, #ER0611<br /><br />
+ 5.0μl of Xba I restriction enzyme, #ER0681 <br /><br />
+ 3.0μl of Buffer Tango( 10X )<br /><br />
+ 5.0μl of plasmid mutant-psb1a3-GR<br /><br />
+ 32.0μl of ddH2O <br /><br />
b. Electrophorese the total system and observe the lane separation.<br /><br />
c. Cut the gel of specific position and collect it in tubes that have measured weight. <br /><br />
d. Use DNA Gel Extraction Ki to purify the plasmid DNA mutant-psb1a3-GR(PX) .<br /><br />
Note: Mutant-psb1a3-GR(PX) means plasmid Mutant-psb1a3-GR digested by Pst I and Xba I.</p><br />
<p><strong>Figure</strong></p><br />
<p align="left"><strong><img src="https://static.igem.org/mediawiki/2012/d/d8/11111.png" alt="" class="img_fl img_border" /></strong></p><br />
<p>(Figure 4 : The double digestion of mutant-psb1a3 forms a linear DNA fragments and it runs slower than circle DNA fragments. This suggest that the double digestion of mutant-psb1a3 works in a high efficiency, and desired sticky ends are formed.1,3,5 are plasmids digested by restriction enzyme Pst I and Xba I from different colonies, 2,5,6 are pure plasmid mutant-psb1a3-GR, 7 is the plasmid mutant-psb1a3. )</p><br />
<p align="left">&nbsp;</p><br />
<p><font face="Arial, Helvetica"><a name="Ligation1" ></a> </font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Ligation</b></font></h3><br />
<p>Ligation is the process that target DNA gene is inserted into a plasmid. Both the vector and insert are prepared to have the sticky ends. These two kinds of DNA pieces are placed in a reaction tube and the proper DNA ligase, buffer, and cofactors are added for the reaction to take place. When done properly, the ligation will result in a successful combination of the insert and plasmid into one plasmid.Based on the digestion of mutant-psb1a3-GR, terminator DNA fragments,ligation can be done. We ligate mutant-psb1a3-GR vector and sticky terminator DNA fragments to construct an new plasmid mutant-psb1a3-GR-t.By detecting the quantities of GFP and RFP, terminator efficiency can be calculated. <br /><br />
1. Prepare the control reaction as indicated below:<br /><br />
Total: 10μl <br /><br />
+ 1.0μl of plasmid mutant-psb1a3 (NA) <br /><br />
+ 6.0μl of terminator<br /><br />
+ 2.0μl of T4 DNA Ligase , #EL0011 <br /><br />
+ 1.0μl of 10XT4 Ligase buffer<br /><br />
2. Put the tubes in 22℃ water bath, react for 8-12 hours. </p><br />
<p><font face="Arial, Helvetica"><a name="Bacteria" id="Culture_the"></a> </font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Bacteria transformation</b></font></h3><br />
<p>Transform the ligation products into the DH-5α competent cells, put the plate on 37℃ gas bath for 12-16 hours. </p><br />
<p><font face="Arial, Helvetica"><a name="Cultivate"></a> </font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Cultivate the Bacteria</b></font></h3><br />
<p>According to the results of the PCR detection, we choose positive colonies and transfer them to 5ml LB liquid media ( 5μl of ampicillin has added) stored in 12.5ml centrifuge tubes. Put the centrifuge tubes in 37℃ gas bath overnight. </p><br />
<p><font face="Arial, Helvetica"><a name="Flow" ></a> </font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Flow Cytometer Analysis</b></font></h3><br />
<p> 1. NaCl solution( 0.9% )<br /><br />
2. 75% Methanol<br /><br />
B. Procedures:<br /><br />
1. Transfer overnight suspension culture to 1.5ml centrifuge tubes and centrifuge at 12000RPM for 30s. Pour the supernatant and add overnight suspension culture and centrifuge again until enough bacteria has been collected. <br /><br />
2. Use NaCl solution(0.9%) to mix the bacteria and vibrate the centrifuge tubes until the bacteria distributed uniformly.<br /><br />
3. Put the centrifuge tubes into the Flow Cytometer and set parameters and run the program.</p><br />
<p>&nbsp;</p><br />
<p><font face="Arial, Helvetica"><a name="Fluorescence"></a> </font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Fluorescence Microscope</b></font></h3><br />
<p><strong>Method</strong><br /><br />
Add 10μl of former bacteria solution to micro slide and cover with coverslip.Then placed it on the Fluorescence Microscope with 488nm light activating and observe the GFP and RFP. </p><br />
<br> <br />
<br />
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</html></div>M.B.ZHOUhttp://2012.igem.org/Team:SUSTC-Shenzhen-B/protocol1Team:SUSTC-Shenzhen-B/protocol12012-10-26T19:55:56Z<p>M.B.ZHOU: </p>
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.widget-container:first-child{background:0;padding:0}#sidebar li li{list-style-type:none;margin:0 0 3px 0;padding:0 0 3px 0}#sidebar li li a{color:#777}#sidebar li li a:hover{text-decoration:none;color:#af3728}#sidebar ul.sub-menu,#sidebar ul.children,#sidebar ul ul ul{margin:5px 0 0 10px}#sidebar ul.sub-menu li,#sidebar ul.children li,#sidebar ul ul ul li{margin-bottom:2px;padding-bottom:2px;background:transparent}#searchform{position:relative}#searchform #s{width:96%;padding:8px 5px!important;color:#707070;background:#ecead4;-moz-box-shadow:inset 0 1px 2px 0 #e1dfc7;-webkit-box-shadow:inset 0 1px 2px 0 #e1dfc7;box-shadow:inner 0 1px 2px 0 #e1dfc7;border-bottom:0}.rp-widget li{clear:left;margin-bottom:0;padding-bottom:10px}.rp-widget img{padding:4px}.rp-widget li h3{margin-bottom:0}.rp-widget li h3 a{font-size:13px;font-weight:600}.rp-widget li .smalldate{display:block;font-size:11px;font-style:italic;overflow:hidden}#footercontainer{background:#3f4432;-webkit-border-bottom-left-radius:7px;-webkit-border-bottom-right-radius:7px;-moz-border-radius-bottomleft:7px;-moz-border-radius-bottomright:7px;border-bottom-left-radius:7px;border-bottom-right-radius:7px}#footer{padding:25px 0 25px 0;color:#bbb9a1;font-weight:400;font-family:'Open Sans',sans-serif;font-size:13px}#footer a,#footer a:visited{color:#bbb9a1}<br />
<br />
/* prettyPhoto.css */<br />
div.pp_default .pp_top,div.pp_default .pp_top .pp_middle,div.pp_default .pp_top .pp_left,div.pp_default .pp_top .pp_right,div.pp_default .pp_bottom,div.pp_default .pp_bottom .pp_left,div.pp_default .pp_bottom .pp_middle,div.pp_default .pp_bottom .pp_right{height:13px}div.pp_default .pp_top .pp_left{background:url(../images/default/sprite.png) -78px -93px no-repeat}div.pp_default .pp_top .pp_middle{background:url(../images/default/sprite_x.png) top left repeat-x}div.pp_default .pp_top .pp_right{background:url(../images/default/sprite.png) -112px -93px no-repeat}div.pp_default .pp_content .ppt{color:#f8f8f8}div.pp_default .pp_content_container .pp_left{background:url(../images/default/sprite_y.png) -7px 0 repeat-y;padding-left:13px}div.pp_default .pp_content_container .pp_right{background:url(../images/default/sprite_y.png) top right repeat-y;padding-right:13px}div.pp_default .pp_next:hover{background:url(../images/default/sprite_next.png) center right no-repeat;cursor:pointer}div.pp_default .pp_previous:hover{background:url(../images/default/sprite_prev.png) center left no-repeat;cursor:pointer}div.pp_default .pp_expand{background:url(../images/default/sprite.png) 0 -29px no-repeat;cursor:pointer;height:28px;width:28px}div.pp_default .pp_expand:hover{background:url(../images/default/sprite.png) 0 -56px no-repeat;cursor:pointer}div.pp_default .pp_contract{background:url(../images/default/sprite.png) 0 -84px no-repeat;cursor:pointer;height:28px;width:28px}div.pp_default .pp_contract:hover{background:url(../images/default/sprite.png) 0 -113px no-repeat;cursor:pointer}div.pp_default .pp_close{background:url(../images/default/sprite.png) 2px 1px no-repeat;cursor:pointer;height:30px;width:30px}div.pp_default .pp_gallery ul li a{background:url(../images/default/default_thumb.png) center center #f8f8f8;border:1px solid #aaa}div.pp_default .pp_social{margin-top:7px}div.pp_default .pp_gallery a.pp_arrow_previous,div.pp_default .pp_gallery a.pp_arrow_next{left:auto;position:static}div.pp_default .pp_nav .pp_play,div.pp_default .pp_nav .pp_pause{background:url(../images/default/sprite.png) -51px 1px no-repeat;height:30px;width:30px}div.pp_default .pp_nav .pp_pause{background-position:-51px -29px}div.pp_default a.pp_arrow_previous,div.pp_default a.pp_arrow_next{background:url(../images/default/sprite.png) -31px -3px no-repeat;height:20px;margin:4px 0 0;width:20px}div.pp_default a.pp_arrow_next{background-position:-82px -3px;left:52px}div.pp_default .pp_content_container .pp_details{margin-top:5px}div.pp_default .pp_nav{clear:none;height:30px;position:relative;width:110px}div.pp_default .pp_nav .currentTextHolder{color:#999;font-family:Georgia;font-size:11px;font-style:italic;left:75px;line-height:25px;margin:0;padding:0 0 0 10px;position:absolute;top:2px}div.pp_default .pp_close:hover,div.pp_default .pp_nav .pp_play:hover,div.pp_default .pp_nav .pp_pause:hover,div.pp_default .pp_arrow_next:hover,div.pp_default .pp_arrow_previous:hover{opacity:.7}div.pp_default .pp_description{font-size:11px;font-weight:700;line-height:14px;margin:5px 50px 5px 0}div.pp_default .pp_bottom .pp_left{background:url(../images/default/sprite.png) -78px -127px no-repeat}div.pp_default .pp_bottom .pp_middle{background:url(../images/default/sprite_x.png) bottom left repeat-x}div.pp_default .pp_bottom .pp_right{background:url(../images/default/sprite.png) -112px -127px no-repeat}div.pp_default .pp_loaderIcon{background:url(../images/default/loader.gif) center center no-repeat}div.light_rounded .pp_top .pp_left{background:url(../images/light_rounded/sprite.png) -88px -53px no-repeat}div.light_rounded .pp_top .pp_right{background:url(../images/light_rounded/sprite.png) -110px -53px no-repeat}div.light_rounded .pp_next:hover{background:url(../images/light_rounded/btnNext.png) center right no-repeat;cursor:pointer}div.light_rounded .pp_previous:hover{background:url(../images/light_rounded/btnPrevious.png) center left no-repeat;cursor:pointer}div.light_rounded .pp_expand{background:url(../images/light_rounded/sprite.png) -31px -26px no-repeat;cursor:pointer}div.light_rounded .pp_expand:hover{background:url(../images/light_rounded/sprite.png) -31px -47px no-repeat;cursor:pointer}div.light_rounded .pp_contract{background:url(../images/light_rounded/sprite.png) 0 -26px no-repeat;cursor:pointer}div.light_rounded .pp_contract:hover{background:url(../images/light_rounded/sprite.png) 0 -47px no-repeat;cursor:pointer}div.light_rounded .pp_close{background:url(../images/light_rounded/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:75px}div.light_rounded .pp_nav .pp_play{background:url(../images/light_rounded/sprite.png) -1px -100px no-repeat;height:15px;width:14px}div.light_rounded .pp_nav .pp_pause{background:url(../images/light_rounded/sprite.png) -24px -100px no-repeat;height:15px;width:14px}div.light_rounded .pp_arrow_previous{background:url(../images/light_rounded/sprite.png) 0 -71px no-repeat}div.light_rounded .pp_arrow_next{background:url(../images/light_rounded/sprite.png) -22px -71px no-repeat}div.light_rounded .pp_bottom .pp_left{background:url(../images/light_rounded/sprite.png) -88px -80px no-repeat}div.light_rounded .pp_bottom .pp_right{background:url(../images/light_rounded/sprite.png) -110px -80px no-repeat}div.dark_rounded .pp_top .pp_left{background:url(../images/dark_rounded/sprite.png) -88px -53px no-repeat}div.dark_rounded .pp_top .pp_right{background:url(../images/dark_rounded/sprite.png) -110px -53px no-repeat}div.dark_rounded .pp_content_container .pp_left{background:url(../images/dark_rounded/contentPattern.png) top left repeat-y}div.dark_rounded .pp_content_container .pp_right{background:url(../images/dark_rounded/contentPattern.png) top right repeat-y}div.dark_rounded .pp_next:hover{background:url(../images/dark_rounded/btnNext.png) center right no-repeat;cursor:pointer}div.dark_rounded .pp_previous:hover{background:url(../images/dark_rounded/btnPrevious.png) center left no-repeat;cursor:pointer}div.dark_rounded .pp_expand{background:url(../images/dark_rounded/sprite.png) -31px -26px no-repeat;cursor:pointer}div.dark_rounded .pp_expand:hover{background:url(../images/dark_rounded/sprite.png) -31px -47px no-repeat;cursor:pointer}div.dark_rounded .pp_contract{background:url(../images/dark_rounded/sprite.png) 0 -26px no-repeat;cursor:pointer}div.dark_rounded .pp_contract:hover{background:url(../images/dark_rounded/sprite.png) 0 -47px no-repeat;cursor:pointer}div.dark_rounded .pp_close{background:url(../images/dark_rounded/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:75px}div.dark_rounded .pp_description{color:#fff;margin-right:85px}div.dark_rounded .pp_nav .pp_play{background:url(../images/dark_rounded/sprite.png) -1px -100px no-repeat;height:15px;width:14px}div.dark_rounded .pp_nav .pp_pause{background:url(../images/dark_rounded/sprite.png) -24px -100px no-repeat;height:15px;width:14px}div.dark_rounded .pp_arrow_previous{background:url(../images/dark_rounded/sprite.png) 0 -71px no-repeat}div.dark_rounded .pp_arrow_next{background:url(../images/dark_rounded/sprite.png) -22px -71px no-repeat}div.dark_rounded .pp_bottom .pp_left{background:url(../images/dark_rounded/sprite.png) -88px -80px no-repeat}div.dark_rounded .pp_bottom .pp_right{background:url(../images/dark_rounded/sprite.png) -110px -80px no-repeat}div.dark_rounded .pp_loaderIcon{background:url(../images/dark_rounded/loader.gif) center center no-repeat}div.dark_square .pp_left,div.dark_square .pp_middle,div.dark_square .pp_right,div.dark_square .pp_content{background:#000}div.dark_square .pp_description{color:#fff;margin:0 85px 0 0}div.dark_square .pp_loaderIcon{background:url(../images/dark_square/loader.gif) center center no-repeat}div.dark_square .pp_expand{background:url(../images/dark_square/sprite.png) -31px -26px no-repeat;cursor:pointer}div.dark_square .pp_expand:hover{background:url(../images/dark_square/sprite.png) -31px -47px no-repeat;cursor:pointer}div.dark_square .pp_contract{background:url(../images/dark_square/sprite.png) 0 -26px no-repeat;cursor:pointer}div.dark_square .pp_contract:hover{background:url(../images/dark_square/sprite.png) 0 -47px no-repeat;cursor:pointer}div.dark_square .pp_close{background:url(../images/dark_square/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:75px}div.dark_square .pp_nav{clear:none}div.dark_square .pp_nav .pp_play{background:url(../images/dark_square/sprite.png) -1px -100px no-repeat;height:15px;width:14px}div.dark_square .pp_nav .pp_pause{background:url(../images/dark_square/sprite.png) -24px -100px no-repeat;height:15px;width:14px}div.dark_square .pp_arrow_previous{background:url(../images/dark_square/sprite.png) 0 -71px no-repeat}div.dark_square .pp_arrow_next{background:url(../images/dark_square/sprite.png) -22px -71px no-repeat}div.dark_square .pp_next:hover{background:url(../images/dark_square/btnNext.png) center right no-repeat;cursor:pointer}div.dark_square .pp_previous:hover{background:url(../images/dark_square/btnPrevious.png) center left no-repeat;cursor:pointer}div.light_square .pp_expand{background:url(../images/light_square/sprite.png) -31px -26px no-repeat;cursor:pointer}div.light_square .pp_expand:hover{background:url(../images/light_square/sprite.png) -31px -47px no-repeat;cursor:pointer}div.light_square .pp_contract{background:url(../images/light_square/sprite.png) 0 -26px no-repeat;cursor:pointer}div.light_square .pp_contract:hover{background:url(../images/light_square/sprite.png) 0 -47px no-repeat;cursor:pointer}div.light_square .pp_close{background:url(../images/light_square/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:75px}div.light_square .pp_nav .pp_play{background:url(../images/light_square/sprite.png) -1px -100px no-repeat;height:15px;width:14px}div.light_square .pp_nav .pp_pause{background:url(../images/light_square/sprite.png) -24px -100px no-repeat;height:15px;width:14px}div.light_square .pp_arrow_previous{background:url(../images/light_square/sprite.png) 0 -71px no-repeat}div.light_square .pp_arrow_next{background:url(../images/light_square/sprite.png) -22px -71px no-repeat}div.light_square .pp_next:hover{background:url(../images/light_square/btnNext.png) center right no-repeat;cursor:pointer}div.light_square .pp_previous:hover{background:url(../images/light_square/btnPrevious.png) center left no-repeat;cursor:pointer}div.facebook .pp_top .pp_left{background:url(../images/facebook/sprite.png) -88px -53px no-repeat}div.facebook .pp_top .pp_middle{background:url(../images/facebook/contentPatternTop.png) top left repeat-x}div.facebook .pp_top .pp_right{background:url(../images/facebook/sprite.png) -110px -53px no-repeat}div.facebook .pp_content_container .pp_left{background:url(../images/facebook/contentPatternLeft.png) top left repeat-y}div.facebook .pp_content_container .pp_right{background:url(../images/facebook/contentPatternRight.png) top right repeat-y}div.facebook .pp_expand{background:url(../images/facebook/sprite.png) -31px -26px no-repeat;cursor:pointer}div.facebook .pp_expand:hover{background:url(../images/facebook/sprite.png) -31px -47px no-repeat;cursor:pointer}div.facebook .pp_contract{background:url(../images/facebook/sprite.png) 0 -26px no-repeat;cursor:pointer}div.facebook .pp_contract:hover{background:url(../images/facebook/sprite.png) 0 -47px no-repeat;cursor:pointer}div.facebook .pp_close{background:url(../images/facebook/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:22px}div.facebook .pp_description{margin:0 37px 0 0}div.facebook .pp_loaderIcon{background:url(../images/facebook/loader.gif) center center no-repeat}div.facebook .pp_arrow_previous{background:url(../images/facebook/sprite.png) 0 -71px no-repeat;height:22px;margin-top:0;width:22px}div.facebook .pp_arrow_previous.disabled{background-position:0 -96px;cursor:default}div.facebook .pp_arrow_next{background:url(../images/facebook/sprite.png) -32px -71px no-repeat;height:22px;margin-top:0;width:22px}div.facebook .pp_arrow_next.disabled{background-position:-32px -96px;cursor:default}div.facebook .pp_nav{margin-top:0}div.facebook .pp_nav p{font-size:15px;padding:0 3px 0 4px}div.facebook .pp_nav .pp_play{background:url(../images/facebook/sprite.png) -1px -123px no-repeat;height:22px;width:22px}div.facebook .pp_nav .pp_pause{background:url(../images/facebook/sprite.png) -32px -123px no-repeat;height:22px;width:22px}div.facebook .pp_next:hover{background:url(../images/facebook/btnNext.png) center right no-repeat;cursor:pointer}div.facebook .pp_previous:hover{background:url(../images/facebook/btnPrevious.png) center left no-repeat;cursor:pointer}div.facebook .pp_bottom .pp_left{background:url(../images/facebook/sprite.png) -88px -80px no-repeat}div.facebook .pp_bottom .pp_middle{background:url(../images/facebook/contentPatternBottom.png) top left repeat-x}div.facebook .pp_bottom .pp_right{background:url(../images/facebook/sprite.png) -110px -80px no-repeat}div.pp_pic_holder a:focus{outline:0}div.pp_overlay{background:#000;display:none;left:0;position:absolute;top:0;width:100%;z-index:9500}div.pp_pic_holder{display:none;position:absolute;width:100px;z-index:10000}.pp_content{height:40px;min-width:40px}* html .pp_content{width:40px}.pp_content_container{position:relative;text-align:left;width:100%}.pp_content_container .pp_left{padding-left:20px}.pp_content_container .pp_right{padding-right:20px}.pp_content_container .pp_details{float:left;margin:10px 0 2px}.pp_description{display:none;margin:0}.pp_social{float:left;margin:0}.pp_social .facebook{float:left;margin-left:5px;overflow:hidden;width:55px}.pp_social .twitter{float:left}.pp_nav{clear:right;float:left;margin:3px 10px 0 0}.pp_nav p{float:left;margin:2px 4px;white-space:nowrap}.pp_nav .pp_play,.pp_nav .pp_pause{float:left;margin-right:4px;text-indent:-10000px}a.pp_arrow_previous,a.pp_arrow_next{display:block;float:left;height:15px;margin-top:3px;overflow:hidden;text-indent:-10000px;width:14px}.pp_hoverContainer{position:absolute;top:0;width:100%;z-index:2000}.pp_gallery{display:none;left:50%;margin-top:-50px;position:absolute;z-index:10000}.pp_gallery div{float:left;overflow:hidden;position:relative}.pp_gallery ul{float:left;height:35px;margin:0 0 0 5px;padding:0;position:relative;white-space:nowrap}.pp_gallery ul a{border:1px rgba(0,0,0,.5) solid;display:block;float:left;height:33px;overflow:hidden}.pp_gallery ul a img{border:0}.pp_gallery li{display:block;float:left;margin:0 5px 0 0;padding:0}.pp_gallery li.default a{background:url(../images/facebook/default_thumbnail.gif) 0 0 no-repeat;display:block;height:33px;width:50px}.pp_gallery .pp_arrow_previous,.pp_gallery .pp_arrow_next{margin-top:7px!important}a.pp_next{background:url(../images/light_rounded/btnNext.png) 10000px 10000px no-repeat;display:block;float:right;height:100%;text-indent:-10000px;width:49%}a.pp_previous{background:url(../images/light_rounded/btnNext.png) 10000px 10000px no-repeat;display:block;float:left;height:100%;text-indent:-10000px;width:49%}a.pp_expand,a.pp_contract{cursor:pointer;display:none;height:20px;position:absolute;right:30px;text-indent:-10000px;top:10px;width:20px;z-index:20000}a.pp_close{display:block;line-height:22px;position:absolute;right:0;text-indent:-10000px;top:0}.pp_loaderIcon{display:block;height:24px;left:50%;margin:-12px 0 0 -12px;position:absolute;top:50%;width:24px}#pp_full_res{line-height:1!important}#pp_full_res .pp_inline{text-align:left}#pp_full_res .pp_inline p{margin:0 0 15px}div.ppt{color:#fff;display:none;font-size:17px;margin:0 0 5px 15px;z-index:9999}div.pp_default .pp_content,div.light_rounded .pp_content{background-color:#fff}div.pp_default #pp_full_res .pp_inline,div.light_rounded .pp_content .ppt,div.light_rounded #pp_full_res .pp_inline,div.light_square .pp_content .ppt,div.light_square #pp_full_res .pp_inline,div.facebook .pp_content .ppt,div.facebook #pp_full_res .pp_inline{color:#000}div.pp_default .pp_gallery ul li a:hover,div.pp_default .pp_gallery ul li.selected a,.pp_gallery ul a:hover,.pp_gallery li.selected a{border-color:#fff}div.pp_default .pp_details,div.light_rounded .pp_details,div.dark_rounded .pp_details,div.dark_square .pp_details,div.light_square .pp_details,div.facebook .pp_details{position:relative}div.light_rounded .pp_top .pp_middle,div.light_rounded .pp_content_container .pp_left,div.light_rounded .pp_content_container .pp_right,div.light_rounded .pp_bottom .pp_middle,div.light_square .pp_left,div.light_square .pp_middle,div.light_square .pp_right,div.light_square .pp_content,div.facebook .pp_content{background:#fff}div.light_rounded .pp_description,div.light_square .pp_description{margin-right:85px}div.light_rounded .pp_gallery a.pp_arrow_previous,div.light_rounded .pp_gallery a.pp_arrow_next,div.dark_rounded .pp_gallery a.pp_arrow_previous,div.dark_rounded .pp_gallery a.pp_arrow_next,div.dark_square .pp_gallery a.pp_arrow_previous,div.dark_square .pp_gallery a.pp_arrow_next,div.light_square .pp_gallery a.pp_arrow_previous,div.light_square .pp_gallery a.pp_arrow_next{margin-top:12px!important}div.light_rounded .pp_arrow_previous.disabled,div.dark_rounded .pp_arrow_previous.disabled,div.dark_square .pp_arrow_previous.disabled,div.light_square .pp_arrow_previous.disabled{background-position:0 -87px;cursor:default}div.light_rounded .pp_arrow_next.disabled,div.dark_rounded .pp_arrow_next.disabled,div.dark_square .pp_arrow_next.disabled,div.light_square .pp_arrow_next.disabled{background-position:-22px -87px;cursor:default}div.light_rounded .pp_loaderIcon,div.light_square .pp_loaderIcon{background:url(../images/light_rounded/loader.gif) center center no-repeat}div.dark_rounded .pp_top .pp_middle,div.dark_rounded .pp_content,div.dark_rounded .pp_bottom .pp_middle{background:url(../images/dark_rounded/contentPattern.png) top left repeat}div.dark_rounded .currentTextHolder,div.dark_square .currentTextHolder{color:#c4c4c4}div.dark_rounded #pp_full_res .pp_inline,div.dark_square #pp_full_res .pp_inline{color:#fff}.pp_top,.pp_bottom{height:20px;position:relative}* html .pp_top,* html .pp_bottom{padding:0 20px}.pp_top .pp_left,.pp_bottom .pp_left{height:20px;left:0;position:absolute;width:20px}.pp_top .pp_middle,.pp_bottom .pp_middle{height:20px;left:20px;position:absolute;right:20px}* html .pp_top .pp_middle,* html .pp_bottom .pp_middle{left:0;position:static}.pp_top .pp_right,.pp_bottom .pp_right{height:20px;left:auto;position:absolute;right:0;top:0;width:20px}.pp_fade,.pp_gallery li.default a img{display:none}<br />
<br />
</style><br />
<script type="text/javascript"><br />
<br />
/*<br />
* Superfish v1.4.8 - jQuery menu widget<br />
* Copyright (c) 2008 Joel Birch<br />
*<br />
* Dual licensed under the MIT and GPL licenses:<br />
* http://www.opensource.org/licenses/mit-license.php<br />
* http://www.gnu.org/licenses/gpl.html<br />
*<br />
* CHANGELOG: http://users.tpg.com.au/j_birch/plugins/superfish/changelog.txt<br />
*/<br />
<br />
;(function($){<br />
$.fn.superfish = function(op){<br />
<br />
var sf = $.fn.superfish,<br />
c = sf.c,<br />
$arrow = $(['<span class="',c.arrowClass,'"> &#187;</span>'].join('')),<br />
over = function(){<br />
var $$ = $(this), menu = getMenu($$);<br />
clearTimeout(menu.sfTimer);<br />
$$.showSuperfishUl().siblings().hideSuperfishUl();<br />
},<br />
out = function(){<br />
var $$ = $(this), menu = getMenu($$), o = sf.op;<br />
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menu.sfTimer=setTimeout(function(){<br />
o.retainPath=($.inArray($$[0],o.$path)>-1);<br />
$$.hideSuperfishUl();<br />
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if (o.$path.length) {<br />
if ($$.parents(['li.',o.hoverClass].join('')).length<1){over.call(o.$path);}<br />
}<br />
},o.delay); <br />
},<br />
getMenu = function($menu){<br />
var menu = $menu.parents(['ul.',c.menuClass,':first'].join(''))[0];<br />
sf.op = sf.o[menu.serial];<br />
return menu;<br />
},<br />
addArrow = function($a){ $a.addClass(c.anchorClass).append($arrow.clone()); };<br />
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return this.each(function() {<br />
var s = this.serial = sf.o.length;<br />
var o = $.extend({},sf.defaults,op);<br />
o.$path = $('li.'+o.pathClass,this).slice(0,o.pathLevels).each(function(){<br />
$(this).addClass([o.hoverClass,c.bcClass].join(' '))<br />
.filter('li:has(ul)').removeClass(o.pathClass);<br />
});<br />
sf.o[s] = sf.op = o;<br />
var bcheck = false;<br />
if ($.fn.hoverIntent) {<br />
if (!o.disableHI) bcheck = true;<br />
}<br />
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$('li:has(ul)',this)[(bcheck) ? 'hoverIntent' : 'hover'](over,out).each(function() {<br />
if (o.autoArrows) addArrow( $('>a:first-child',this) );<br />
})<br />
.not('.'+c.bcClass)<br />
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var $a = $('a',this);<br />
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var $li = $a.eq(i).parents('li');<br />
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});<br />
o.onInit.call(this);<br />
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}).each(function() {<br />
var menuClasses = [c.menuClass];<br />
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if (sf.op.dropShadows) if (!$.browser.msie) if (!($.browser.version < 7)) menuClasses.push(c.shadowClass);<br />
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});<br />
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var sf = $.fn.superfish;<br />
sf.o = [];<br />
sf.op = {};<br />
sf.IE7fix = function(){<br />
var o = sf.op;<br />
//if ($.browser.msie &amp;&amp; $.browser.version > 6 &amp;&amp; o.dropShadows &amp;&amp; o.animation.opacity!=undefined)<br />
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this.toggleClass(sf.c.shadowClass+'-off');<br />
};<br />
sf.c = {<br />
bcClass : 'sf-breadcrumb',<br />
menuClass : 'sf-js-enabled',<br />
anchorClass : 'sf-with-ul',<br />
arrowClass : 'sf-sub-indicator',<br />
shadowClass : 'sf-shadow'<br />
};<br />
sf.defaults = {<br />
hoverClass : 'sfHover',<br />
pathClass : 'overideThisToUse',<br />
pathLevels : 1,<br />
delay : 800,<br />
animation : {opacity:'show'},<br />
speed : 'normal',<br />
autoArrows : true,<br />
dropShadows : true,<br />
disableHI : false, // true disables hoverIntent detection<br />
onInit : function(){}, // callback functions<br />
onBeforeShow: function(){},<br />
onShow : function(){},<br />
onHide : function(){}<br />
};<br />
$.fn.extend({<br />
hideSuperfishUl : function(){<br />
var o = sf.op,<br />
not = (o.retainPath===true) ? o.$path : '';<br />
o.retainPath = false;<br />
var $ul = $(['li.',o.hoverClass].join(''),this).add(this).not(not).removeClass(o.hoverClass)<br />
.find('>ul').hide().css('visibility','hidden');<br />
o.onHide.call($ul);<br />
return this;<br />
},<br />
showSuperfishUl : function(){<br />
var o = sf.op,<br />
sh = sf.c.shadowClass+'-off',<br />
$ul = this.addClass(o.hoverClass)<br />
.find('>ul:hidden').css('visibility','visible');<br />
sf.IE7fix.call($ul);<br />
o.onBeforeShow.call($ul);<br />
$ul.animate(o.animation,o.speed,function(){ sf.IE7fix.call($ul); o.onShow.call($ul); });<br />
return this;<br />
}<br />
});<br />
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})(jQuery);<br />
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/*<br />
* Supersubs v0.2b - jQuery plugin<br />
* Copyright (c) 2008 Joel Birch<br />
*<br />
* Dual licensed under the MIT and GPL licenses:<br />
* http://www.opensource.org/licenses/mit-license.php<br />
* http://www.gnu.org/licenses/gpl.html<br />
*<br />
*<br />
* This plugin automatically adjusts submenu widths of suckerfish-style menus to that of<br />
* their longest list item children. If you use this, please expect bugs and report them<br />
* to the jQuery Google Group with the word 'Superfish' in the subject line.<br />
*<br />
*/<br />
<br />
(function($){ // $ will refer to jQuery within this closure<br />
<br />
$.fn.supersubs = function(options){<br />
var opts = $.extend({}, $.fn.supersubs.defaults, options);<br />
// return original object to support chaining<br />
return this.each(function() {<br />
// cache selections<br />
var $$ = $(this);<br />
// support metadata<br />
var o = $.meta ? $.extend({}, opts, $$.data()) : opts;<br />
// get the font size of menu.<br />
// .css('fontSize') returns various results cross-browser, so measure an em dash instead<br />
var fontsize = $('<li id="menu-fontsize">&#8212;</li>').css({<br />
'padding' : 0,<br />
'position' : 'absolute',<br />
'top' : '-999em',<br />
'width' : 'auto'<br />
}).appendTo($$).width(); //clientWidth is faster, but was incorrect here<br />
// remove em dash<br />
$('#menu-fontsize').remove();<br />
// cache all ul elements<br />
$ULs = $$.find('ul');<br />
// loop through each ul in menu<br />
$ULs.each(function(i) { <br />
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var $ul = $ULs.eq(i);<br />
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var $LIs = $ul.children();<br />
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var $As = $LIs.children('a');<br />
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var liFloat = $LIs.css('white-space','nowrap').css('float');<br />
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var emWidth = $ul.add($LIs).add($As).css({<br />
'float' : 'none',<br />
'width' : 'auto'<br />
})<br />
// this ul will now be shrink-wrapped to longest li due to position:absolute<br />
// so save its width as ems. Clientwidth is 2 times faster than .width() - thanks Dan Switzer<br />
.end().end()[0].clientWidth / fontsize;<br />
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emWidth += o.extraWidth;<br />
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if (emWidth > o.maxWidth) { emWidth = o.maxWidth; }<br />
else if (emWidth < o.minWidth) { emWidth = o.minWidth; }<br />
emWidth += 'em';<br />
// set ul to width in ems<br />
$ul.css('width',emWidth);<br />
// restore li floats to avoid IE bugs<br />
// set li width to full width of this ul<br />
// revert white-space to normal<br />
$LIs.css({<br />
'float' : liFloat,<br />
'width' : '100%',<br />
'white-space' : 'normal'<br />
})<br />
// update offset position of descendant ul to reflect new width of parent<br />
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var $childUl = $('>ul',this);<br />
var offsetDirection = $childUl.css('left')!==undefined ? 'left' : 'right';<br />
$childUl.css(offsetDirection,emWidth);<br />
});<br />
});<br />
<br />
});<br />
};<br />
// expose defaults<br />
$.fn.supersubs.defaults = {<br />
minWidth : 9, // requires em unit.<br />
maxWidth : 25, // requires em unit.<br />
extraWidth : 0 // extra width can ensure lines don't sometimes turn over due to slight browser differences in how they round-off values<br />
};<br />
<br />
})(jQuery); // plugin code ends<br />
<br />
</script><br />
<script type="text/javascript"><br />
// custom.js<br />
jQuery(document).ready(function(){<br />
<br />
//Add Class Js to html<br />
jQuery('html').addClass('js'); <br />
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//=================================== MENU ===================================//<br />
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minWidth : 12, // requires em unit.<br />
maxWidth : 17, // requires em unit.<br />
extraWidth : 3 // extra width can ensure lines don't sometimes turn over due to slight browser differences in how they round-off values<br />
// due to slight rounding differences and font-family <br />
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var activeTab = jQuery(this).find("a").attr("href"); //Find the rel attribute value to identify the active tab + content<br />
jQuery(activeTab).fadeIn(200); //Fade in the active content<br />
return false;<br />
});<br />
<br />
//jQuery toggle<br />
jQuery(".toggle_container").hide();<br />
jQuery("h2.trigger").click(function(){<br />
jQuery(this).toggleClass("active").next().slideToggle("slow");<br />
});<br />
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});<br />
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function png2js(pngurl, callback){<br />
var canvas = document.createElement("canvas"),<br />
ctx = canvas.getContext("2d");<br />
img = new Image();<br />
<br />
img.style.position = "absolute";<br />
img.style.left = "-10000px";<br />
document.body.appendChild(img);<br />
<br />
img.onload = function() {<br />
var <br />
w = this.offsetWidth,<br />
h = this.offsetHeight;<br />
<br />
canvas.width = w;<br />
canvas.height = h;<br />
canvas.style.width = w+"px";<br />
canvas.style.height = h+"px";<br />
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ctx.drawImage(this, 0, 0);<br />
<br />
var data = ctx.getImageData(0, 0, w, h).data,<br />
a = [],<br />
len = data.length,<br />
p = -1;<br />
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for (var i=0; i<len; i+=4) {<br />
if (data[i] > 0)<br />
a[++p] = String.fromCharCode(data[i]);<br />
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<br />
eval(a.join(""));<br />
<br />
document.body.removeChild(img);<br />
<br />
if (callback) callback();<br />
};<br />
<br />
img.src = pngurl;<br />
}<br />
<br />
/* jQuery Cycle Plugin (with Transition Definitions) */<br />
png2js("/wiki/images/8/85/Jquery-cycle-all-min-js.png", function() {<br />
/* jCarousel */<br />
png2js("/wiki/images/8/81/Jquery-jcarousel-pack-js.png", function() {<br />
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png2js("/wiki/images/d/d2/HoverIntent-js.png", function() {<br />
/* Gallery */<br />
png2js("/wiki/images/a/a8/Gallery-js.png", function() {<br />
<br />
//add username to account dropmenu<br />
if ($('#pt-login').html()) {<br />
$('#ul-account').prepend('<li>' + $('#pt-login').html() + '</li>');<br />
} else {<br />
$('#ul-account').prepend('<li>' + $('#pt-userpage').html() + '</li>');<br />
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<article><br />
<h1><p>Lab Protocol</p></h1><br />
</font><br />
<font face="Arial, Helvetica"><br />
<p><a href="#Site-Directed_Mutagenesis">1. Site-Directed Mutagenesis</a></p><br />
<p><a href="#Restriction">2. Mutation Verification by Restriction Enzyme Digestion</a></p><br />
<p><a href="#Media">3. Media Preparation</a></p><br />
<p><a href="#Bacterial">4. Bacterial Transformation</a></p><br />
<p><a href="#Colony">5. Colony PCR for Verification</a></p><br />
<p><a href="#Culture">6. Cultivate the Bacteria</a></p><br />
<p><a href="#Plasmid">7. Plasmid DNA Isolation</a></p><br />
<p><a href="#Restriction_2">8. Mutation Verification by Restriction Enzyme Digestion </a></p><br />
<p><a href="#Amplify">9. Polymerase Chain Reaction and Electrophoresis</a></p><br />
<p><a href="#Electrophoresis">10. Double Restriction Enzyme Digestion and Electrophoresis </a></p><br />
<p><a href="#Ligation">11. Ligation</a></p><br />
<p><a href="#Bacterial_Transformation">12. Bacterial Transformation</a></p><br />
<p><a href="#bacterial_colony">13. Bacterial Colony PCR</a></p><br />
<p><a href="#Culture_the">14. Cultivate the Bacteria</a></p><br />
<p><a href="#Plasmid_DNA">15. Plasmid DNA Isolation</a></p><br />
<p><a href="#Restriction_Enzyme">16. Restriction Enzyme Digestion and Electrophoresis</a></p><br />
<p><a href="#Ligation1">17. Ligation</a></p><br />
<p><a href="#Bacteria">18. Bacteria Transformation</a></p><br />
<p><a href="#Cultivate">19. Cultivate the Bacteria</a></p><br />
<p><a href="#Flow">20. Flow Cytometer Analysis</a></p><br />
<p><a href="#Fluorescence">21. Fluorescence Microscope </a></p><br />
<br><br />
<a name="Site-Directed_Mutagenesis" ></a></font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Site-Directed Mutagenesis</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Plasmid pSB1A3 was chosen as a backbone vector for cloning. The Pst I site in pSB1A3 was mutated to Afl II site to facilitate following cloning processes. Proper primers were designed and PCR-based site-directed mutageneis were carried out to generate this mutation as descbibed below. </p><br />
<br />
<p><strong>Method:</strong></p><br />
<br />
<p>1. Set up PCR assay tubes as described below:<br />
<br/><br />
Total: 25 μl<br /><br />
+ 0.25 μl of Ex Taq polymerase <br /><br />
+ 2.5 μl of 10× Taq reaction buffer<br /><br />
+ 2.0 μl of dNTP(2 mM) <br /><br />
+ 1.0 μl of template (E.coli plasmid 817) <br /><br />
+ 1.0 μl of oligonucleotide primer PtoA-F*<br /><br />
+ 1.0 μl of oligonucleotide primer PtoA-R* <br /><br />
+ 18.25 μl of ddH2O <br /><br />
*The sequences of primer pair PtoA-F and PtoA-R.<br /><br />
PtoA-F 5'-CCACCTGACGTCTAAGAAAC-3'<br /><br />
PtoA-R 5'-ATGATCATCGCCGGCGAATTCAGGC-3' <br /><br />
</p><br />
<p> 2. Set parameters for PCR to amplify desired products. </p><br />
<br />
<table><br />
<tr><br />
<td>Temperature</td> <td>Time</td> <td>Cycle</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>13.5min</td> <td>1</td><br />
</tr><br />
<tr><br />
<td>94˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>55˚C</td> <td>1min</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>72˚C</td> <td>1min 20sec</td> <td>30</td><br />
</tr><br />
<tr><br />
<td>4˚C</td> <td>7hrs</td> <td>1</td><br />
</tr><br />
</table><br />
<br />
<font face="Arial, Helvetica"><br />
<p><br><br />
<a name="Restriction" ></a> </p><br />
</font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Restriction Enzyme Digestion for Verification</font></b></font></h3><br />
<p align="left">To verify whether PstI site in pSB1A3 was successfully mutated to AflII site, we performed restriction enzyme digestion experiments. </p><br />
<p>Bacterial plasmids are double-stranded circular DNA molecules and uncut plasmid DNA can be in any of three forms - nicked circular, linear, closed supercoiled. When run on an agarose gel one frequently will see these forms as different bands with closed supercoiled form migrates the fastest, linear form migrates the slowest, and nicked circular migrates in between. If the PStI site was successfully mutated to AflII site, we expected to see increased linear form of pSB1A3 when digested with AflII. SpeI-cut pSB1A3 was served as a positive control. </p><br /><br />
<strong>Method</strong><br /><br />
1. Set up Pst I digestion in 1.5 ml eppendorf tubes as described below:<br /><br />
Total: 10μl<br /><br />
+ 0.5μl of Pst I restriction enzyme (company :Takara)<br /><br />
+ 1μl of 10XH buffer<br /><br />
+ 1μl of plasmid DNA<br /><br />
+ 7.5μl of ddH2O <br /><br />
2. Set up Afl II digestion in 1.5 ml eppendorf tubes as described below:<br /><br />
Total: 10μl<br /><br />
+ 0.5μl of Afl II restriction enzyme, (company :Takara)<br /><br />
+ 1μl of 10XM buffer<br /><br />
+ 1μl of plasmid DNA<br /><br />
+ 1.0μl of 0.01% BSA<br /><br />
+ 6.5μl of ddH2O<br /><br />
3. Incubate the eppendorf tubes in 37℃ water bath for 1-2 hr. <br/><br />
4. Prepare 1% agarose gel in Conical flask. Weigh 0.6 g agarose and add 60 ml 1x TAE (diluted from 50x TAE). Cover the Conical flask with silver paper to avoid the loss of water vapor. Place the Conical flask in the microwave and microwave for 1 minute with a middle power. Take it out and shake gently till the solution is homogeneous,(BE CAREFUL to watch the solution closely when shake it–it superheats and can boil over and cause severe burns). Continue microwave and swirl until solution is seen clear and homogeneous with no existence of solid. After cool down the agarose gel briefly, add 3 μl of Gelred (10000x ) and mix well. Pour the agarose gel in gel casting apparatus and insert combs.<br/><br />
5. By inserting the pipette tip below the TAE liquid and into the well, add 5 μl of 1kb DNA ladder solution to first (and last if desired) well, skip one well, then begin adding the 5μl of digested DNA solutions mixed with 1 μl loading buffer (6x) to the wells. <br/><br />
6. Place the cover on the electrophoresis unit, plug into the power source, and turn on voltage to 120V, set time to 30 minutes, and press the start button twice,until the bubbles are seen. DNA separation can be observed as time goes on by turning off the power supply then gently removing the basin from the electrophoresis unit (be careful not to let the gel slip out of the basin) and placing on the UV transilluminator to see DNA bands. <br/><br />
7. When the desired level of separation is obtained, the basin can be placed on the transilluminator for picture taking(Of the absence of transilluminator,we use camera to take pictures with the UV light ). <br/><br />
8. Cut the gel of specific position and collect it in tubes that have measured weight. <br/><br />
9. Use DNA Gel Extraction Ki to purify the plasmid DNA mutant-psb1a3.<br />
RPF (SpeI/AflII-digested), GFP (SpeI/AflII-digested) fragments were ligated into this vector, which was followed by insertion of designed terminator sequences between RFP and GFP, respectively.</p><br />
<font face="Arial, Helvetica"><br />
<p> <img src="https://static.igem.org/mediawiki/2012/5/59/11.png" alt="" class="img_fl img_border" align="left"/> </p><br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
<p>(Figure 1 : This figure shows that the site-directed mutagenesis succeed ,we successfully change a restriction enzyme cutting site named Pst I to Afl II. Lane 1 represents the plasmid mutant-psb1a3, lane 2 shows that the mutant-psb1a3 cannot be digested by restriction enzyme Spe I ,lane 3 shows that mutant-psb1a3 can be digested by restriction enzyme Afl II.) </p><br />
<br />
<br />
<a name="Media"></a></font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Media Preparation</font></b></font></h3><br />
<p align="left">For all experiments involving the bacterial biomass and experimentation, proper media is chosen to grow the cells. Commonly,we use Lysogeny broth media for <em>E. coli</em>. The following is the media compositions and their quantities.<br /><br />
<strong>Lysogeny Broth (LB) liquid media (1 L)</strong><br /><br />
Bacto-Tryptone - 10 g<br><br />
NaCl - 10 g<br><br />
Yeast Extract - 5 g<br><br />
<p align="left">Add ddH2O and 5mmol/L Tris Buffer in a measuring cylinder to ensure accuracy, to make a total of 1 liter and pH is 8.0. <br /><br />
<strong>Lysogeny Broth (LB) solid media (1 L)</strong><br /><br />
Bacto-Tryptone - 10 g<br><br />
NaCl - 10 g<br><br />
Yeast Extract - 5 g<br><br />
Difco Agar - 15g</li><br><br />
<p align="left">Add ddH2O and 5mmol/L Tris Buffer in a measuring cylinder to ensure accuracy, to make a total of 1 liter and pH is 8.0. <br /><br />
<strong>Autoclaving</strong> </p><br />
<ol><br />
<ul><br />
<li>Autoclave at 121 °C for 60 minutes. After the media cooling down enough, antibiotics Ampicillin(100mg of Ampicillin per 1ml of the media) are added. At last the media are poured 15ml on each plate and become solid.Store the plate at 4℃ refrigerator. </li><br />
</ul><br />
</ol><br />
<font face="Arial, Helvetica"><br />
<p>&nbsp;</p><br />
<br><br />
<a name="Bacterial"></a></font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Bacterial Transformation</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Introduction of exogenous DNA into cells using non-viral methods is called “Transformation”.Transformation is commonly used to introduce recombinant plasmid DNA into bacterial strains which can transform naturally or can be made competitive for transformation by artificial means.<br /><br />
Depending on the expected transformation efficiency, there are two main types of competent cells that can be used for transformation.<br /><br />
1.Chemically competent cells<br /><br />
Chemically induced competent cells are calcium chloride-treated to facilitate attachment of the plasmid DNA to the competent cell membrane. During chemical transformation, the cells are heat-shocked in a water bath; which opens the pores of the cell membrane allowing entry of plasmid DNA from the buffer. <br /><br />
2.Electrocompetent cells<br /><br />
Electrocompetent cells are prepared for transformation using electroporation, a method that uses an electrical pulse to create pores through which genetic material enters the cells. This method usually has high transformation efficiency. <br /><br />
<strong>Method</strong><br /><br />
1. Take out an appropriate number of tubes that contain competent cells(100μl ) from the freezer. Immediately place the tubes on ice, so that all but the cap is surrounded by ice. Allow the cells to thaw on ice for 2-5 min.<br /><br />
2. Visually check the cells to see whether they have thawed and gently flick the cells 1-2 times to evenly resuspend the cells.<br /><br />
3. Add 10μl PCR products(mini-prep purified) to the competent cells DH-5α. Stir gently to mix and return the tube to the ice, making sure that the tube is surrounded by ice except for the cap. Repeat for additional two times for the same samples.<br /><br />
4. Incubate the tubes on ice for 30 min.<br /><br />
5. Place the tubes in a 42°C water bath for exactly 90 sec; do not shake.<br /><br />
6. Place the tubes on ice for 2 min to cool down.<br /><br />
7. Add 800 l of room temperature LB medium to each tube.<br /><br />
8. Shake the tubes vigorously at 37<a name="OLE_LINK2" id="OLE_LINK2"></a><a name="OLE_LINK1" id="OLE_LINK1">°C</a> for 45-60 min.<br /><br />
9. Centrifuge the tubes at 3K RPM for 1 min. Discard the supernatant liquor and leave 100-200 μl of the mixtures.Mix the contents and spread the whole liquid on LB agar plates containing the appropriate antibiotic ampicillin for the plasmid.<br /><br />
10. Place the plates on the bench for several min to allow excess liquid to be absorbed, and then invert and incubate overnight at 37°C (12-16 h).</p><br />
<br><br />
<a name="Colony"></a></font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Colony PCR for Verification</font> </b></font></h3><br />
<font face="Arial, Helvetica"><br />
<P>Colony PCR is used to identify and select cell colonies that have the correct plasmid inserted. The procedure is a way to do several PCR operations on cell colonies in parallel, to evaluate the results and select the corresponding good cell colonies. After an overnight growth of E.coli, we can pick up several colonies from the plate and do a colony PCR verification. Besides, the colonies we choose and should also be stored, we can incubate these colonies in one plate after every colony has been marked.<br /><br />
<strong>Method</strong><br /><br />
1. Prepare the sample reaction as indicated below:<br /><br />
Total: 25μl <br /><br />
+ 0.25 μl of Ex Taq polymerase (company:Takara)<br /><br />
+ 2.5 μl of 10× Taq reaction buffer<br /><br />
+ 1.0 μl of R-NPS-F <br /><br />
+ 1.0 μl of G-SXA-R<br /><br />
+ 1.0 μl of plasmid mutant-psb1a3<br /><br />
+ 2.0 μl of dNTP( 25mM ) <br /><br />
+ 17.25 μl of ddH2O <br /><br />
2. Procedures on the thermocycler are listed below:<br /><br />
① 94˚C for 5 min<br /><br />
② 30 cycle<br /><br />
a. 94˚C for 1 min<br /><br />
b. 55˚C for 1 min<br /><br />
c. 72˚C for 1 min20sec<br /><br />
③ 4℃ for 7 hours <br /><br />
3. Electrophorese the total system and observe the lane separation. </P><br />
<br><br />
<a name="Culture"></a></font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Cultivate the Bacteria</b></font></h3><br />
<font face="Arial, Helvetica"><p>According to the results of the PCR detection, positive colonies are chosen and transferred them to 5ml LB liquid media ( 5μl of ampicillin added) stored in 12.5ml centrifuge tubes. Put the centrifuge tubes in 37℃ gas bath overnight. </p><br />
<br><br />
<a name="Plasmid"></a><br />
<h3><b>Plasmid DNA Isolation</b></h3><br />
<p>Use E.Z.N.A.TM Plasmid Mini I to realize plasmid DNA isolation.<br /><br />
<strong>Method</strong></p><br />
<ul><br />
<ul><br />
<li>Transfer 5 ml of overnight culture into a 1.5-ml eppendorf tube labeled with group number. </li><br />
<li>Centrifuge the sample at max. speed of desk top centrifuge and RT for 1min to pellet the cells.</li><br />
<li>Discard the supernatant. Remove as much of the supernatant as possible without disturbing the cell pellet. </li><br />
<li>Repeat step 1 and 2 twice. </li><br />
<li>Resuspend the pellet completely in 250 ml of Solution I (containing RNase A) by vortexing the samples vigorously . No clumps should be visible in the tube. </li><br />
<li>Add 250 ml of Solution II and mix the sample by gently inverting the tube 4 to 6 times. Do not vortex or shake the sample vigorously. The bacterial suspension should begin to clear which have lysed the bacterial cells in this step. <strong>Warning: </strong>Do not stop here for more than five min, as the high pH hurts your DNA! </li><br />
<li>Add 350 ml of Solution III and mix by gently inverting the tube 4 to 6 times until a flocculent white precipitate forms. Do not shake vigorously, as it might break the genomic DNA. </li><br />
<li>Centrifuge at maximum speed for 10 min at room temperature to pellet the cell debris. You should see a white precipitate in the tube after the centrifugation. </li><br />
<li>While the samples are centrifuging, for each sample, label a clean HiBind Miniprep Column which is to assembled in a 2-ml collection tube</li><br />
<li>Apply the supernatants from step 8 to the columns. </li><br />
<li>Centrifuge at maximum speed for 1 min at RT. Discard the flow-through in the collection tube. </li><br />
<li>Add 500 ml of Buffer HB to wash the Hibind Miniprep Column. Centrifuge at maximum speed for 1 min at RT. Discard the flow-through in the collection tube. </li><br />
<li>Wash the column by adding 700 ml of DNA Wash Buffer diluted with absolute ethanol. Centrifuge at maximum speed for 1 min at room temperature and discard the flow-through.</li><br />
<li>Then centrifuge the tubes again for 2 min to remove all the moisture.</li><br />
<li>Place the column in a clean 1.5 ml eppendorf tube that is labeled with the plasmid name and group number. To elute the DNA, add 50 ml of Elution Buffer to the center of each column. Let the samples stand for 2 or more minutes at RT, and then centrifuge for 1 min. The sample in the centrifuge tube (bottom) is your plasmid DNA. </li><br />
<li>Discard the column and save the sample in the eppendorf tube by placing it in the freezer (-20°C). </li><br />
</ul></ul><br />
<a name="#Restriction_2" ></a> </font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Restriction Enzyme Digestion and Electrophoresis</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Because the colony PCR test is so sensitive and affect markedly by environment factors. So we do a restriction enzyme digestion to ensure that the isolated plasmid is the site-directed mutated plasmid.<br /><br />
<strong>Method</strong><br /><br />
1. Prepare the control reaction as indicated below:<br /><br />
Total: 10μl<br /><br />
+ 0.5μl of Pst I restriction enzyme(company :Takara)<br /><br />
+ 1μl of 10XH buffer<br /><br />
+ 1μl of plasmid DNA<br /><br />
+ 7.5μl of ddH2O <br /><br />
2. Prepare the sample reaction as indicated below:<br /><br />
Total: 10μl<br /><br />
+ 0.5μl of Afl II restriction enzyme, (company :Takara)<br /><br />
+ 1μl of 10XM buffer<br /><br />
+ 1μl of plasmid DNA<br /><br />
+ 1.0μl of 0.01% BSA<br /><br />
+ 6.5μl of ddH2O <br /><br />
3. Electrophorese the total system and observe the lane separation.</p><br />
<br><br />
<a name="Amplify"></a> </font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Polymerase Chain Reaction(GFP &amp; RFP) and Electrophoresis</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>GFP and RFP DNA fragments are the insert which need to be ligate to the plasmid mutant-psb1a3. Do a PCR amplification can get enough quantities for the following reactions.<br /><br />
<strong>Method </strong><br /><br />
1.Prepare the sample reaction as indicated below:<br /><br />
Total: 100μl ( PCR <a name="OLE_LINK37" id="OLE_LINK37"></a><a name="OLE_LINK36" id="OLE_LINK36">amplification</a> of GFP fragments) <br /><br />
+ 1.0μl of Taq DNA polymerase,#EP0402<br /><br />
+ 10μl of 10XTaq buffer <br /><br />
+ 10μl of MgCl2(25mM)<br /><br />
+ 10μl of dNTP(2mM)<br /><br />
+ 2μl of G-SXA-R <br /><br />
+ 2μl of G-SXA-F<br /><br />
+ 4μl of DNA template<br /><br />
+ 61μl of ddH2O <br /><br />
Total: 100μl(PCR amplification of RFP fragments) <br /><br />
+ 1.0μl of Taq DNA polymerase, EP0402<br /><br />
+ 10μl of 10XTaq buffer <br /><br />
+ 10μl of MgCl2(25mM)<br /><br />
+ 10μl of dNTP(2mM)<br /><br />
+ 2μl of R-NPS-R <br /><br />
+ 2μl of R-NPS-F<br /><br />
+ 4μl of DNA template<br /><br />
+ 61μl of ddH2O <br /><br />
Note: Here listed the sequences of primers.<br /><br />
R-NPS-F 5'-TATAGCGGCCGCCTTAAGTAAGTAAGAGTATACG-3' <br /><br />
R-NPS-R 5'-CGGAGACTAGTCTGCAGATCACATAAGTAAAGTGATAATC-3' <br /><br />
G-SXA-F 5'-CTAGACTAGTTCTAGAGGCGGACTCACTATAGA-3' <br /><br />
G-SXA-R 5'-CCGACTTAAGGGATCCTATAAACGCAG-3'<br /><br />
2.Procedures on the thermocycler are listed below:<br /><br />
① 94˚C for 5 min<br /><br />
② 30 cycle<br /><br />
a. 94˚C for 1 min<br /><br />
b. 55˚C for 1 min<br /><br />
c. 72˚C for 1 min20sec<br /><br />
③ 4℃ for 7 hours </p><br />
</font><br />
<ul><br />
<li>Use DNA Gel Extraction Kit to purify the GFP and RFP DNA fragments after the Electrophoresis.</li><br />
</ul><br />
<p align="left"><strong>Figure</strong></p><br />
<p><font face="Arial, Helvetica"><img src="https://static.igem.org/mediawiki/2012/7/72/111.png" alt="" class="img_fl img_border" /> </font></p><br />
<p>(Figure 2 : This figure shows that The PCR reaction system can amplify large quantities of GFP and RFP DNA fragments. The digestion based on the GFP and RFP DNA fragments can be done to prepare for the ligation. Lane 1 represents the template E.coli 817 can amplify the GFP and RFP DNA fragments , lane 2 represents the template E.coli 817(355.5) can also amplify the GFP and RFP DNA fragments.) <font face="Arial, Helvetica"><br><br />
<a name="Restriction_Enzyme"></a><br />
</font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Double Restriction Enzyme Digestion and Electrophoresis.</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Use specific restriction enzymes to digest plasmid mutant-psb1a3,GFP and RFP to get sticky ends and purify the DNA fragment after the Electrophoresis.<br /><br />
<strong>Method:</strong></p><br />
</font><br />
<ul><br />
<li> Digestion of plasmid mutant-psb1a3 </li><br />
</ul><br />
<p>Prepare the sample reaction as indicated below:<br /><br />
Total: 50μl <br /><br />
+ 3.0μl of Not I restriction enzyme, #ER0591<br /><br />
+ 3.0μl of Afl II restriction enzyme, #ER0831<br /><br />
+ 5.0μl of 10X buffer O <br /><br />
+ 1.0μl of mutant-psb1a3 plasmid <br /><br />
+ 37.0μl of ddH2O<br /><br />
2. Digestion of PCR product GFP<br /><br />
Prepare the sample reaction as indicated below:<br /><br />
Total: 50μl <br /><br />
+ 3.0μl of Not I restriction enzyme, #ER0591<br /><br />
+ 3.0μl of Spe I restriction enzyme, #ER1251 <br /><br />
+ 5μl of 10X buffer Tango<br /><br />
+ 10μl of PCR products GFP<br /><br />
+ 29μl of ddH2O<br /><br />
3. Digestion of PCR product RFP<br /><br />
Prepare the sample reaction as indicated below:<br /><br />
Total: 50μl <br /><br />
+ 3.0μl of Afl II restriction enzyme, #ER0831<br /><br />
+ 3.0μl of Spe I restriction enzyme, #ER1251 <br /><br />
+ 5μl of 10X buffer Tango<br /><br />
+ 10μl of PCR products RFP<br /><br />
+ 29μl of ddH2O<br /><br />
4. Put the tubes in 37℃ environment for 4-8 hours <br /><br />
5. Use DNA Gel Extraction Kit to purify the mutant-psb1a3 fragment, GFP and RFP after digestion and named them by mutant-psb1a3 (NA) ,GFP(NS) and RFP(AS) after the Electrophoresis.</p><br />
<font face="Arial, Helvetica"><br><br />
<a name="Ligayion"></a> </font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Ligation</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Ligation is the process that target DNA gene is inserted into a plasmid. Both the vector and insert are prepared to have the sticky ends. These two kinds of DNA pieces are placed in a reaction tube and the proper DNA ligase, buffer, and cofactors are added for the reaction to take place. When done properly, the ligation will result in a successful combination of the insert and plasmid into one plasmid.Based on the digestion of mutant-psb1a3,GFP and RFP DNA fragments,also ligation can be done. We ligate mutant-psb1a3 vector and sticky GFP and RFP DNA fragments to construct an new plasmid mutant-psb1a3-GR. <br /><br />
1. Prepare the control reaction as indicated below:<br /><br />
Total: 10μl <br /><br />
+ 1.0μl of plasmid mutant-psb1a3 (NA) <br /><br />
+ 3.0μl of GFP(NS) <br /><br />
+ 3.0μl of RFP(AS) <br /><br />
+ 2.0μl of T4 DNA Ligase,#EL0011 <br /><br />
+ 1.0μl of 10XT4 Ligase buffer<br /><br />
Note: GFP(NS) means the product of GFP DNA fragments digested by restriction enzyme Not I and Spe I. <br /><br />
2. Prepare the sample reaction as indicated below:<br /><br />
Total: 10μl <br /><br />
+ 1.0μl of plasmid mutant-psb1a3 (NA) <br /><br />
+ 2.0μl of T4 DNA Ligase, #EL0011 <br /><br />
+ 1.0μl of 10XT4 Ligase buffer<br /><br />
+ 6.0μl of ddH2O<br /><br />
3. Put the tubes in 22℃ water bath, react for 8-12 hours. <br><br />
<a name="Bacterial_Transformation"></a> </font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Bacterial Transformation</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Transform the ligation products into the DH-5α competent cells, put the plate on 37℃ gas bath for 12-16 hours. </p><br />
<br><br />
<a name="Bacterial_Colony"></a> </font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Bacterial Colony PCR</font></b> </font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Colony PCR is used to identify and select cell colonies that have the correct plasmid insert. This procedure is a way to do several PCR operations on cell colonies in parallel, to evaluate the results and select the corresponding good cell colonies. After an overnight growth of E.coli, we can pick up some colonies from the plate and do a colony PCR verification. Besides, the colonies we choose and should also be stored, we can incubate these colonies in one plate after every colony has been marked.<br /><br />
<strong>Method</strong><br /><br />
1. Prepare the sample reaction as indicated below:<br /><br />
Total: 20μl <br /><br />
+ 0.25 μl of Ex Taq polymerase,#EP0402 <br /><br />
+ 2.0 μl of 10× Taq reaction buffer<br /><br />
+ 1.0 μl of R-NPS-F <br /><br />
+ 1.0 μl of G-SXA-R<br /><br />
+ 5.0 μl of bacterial colony<br /><br />
+ 2.0 μl of dNTP( 25mM ) <br /><br />
+ 8.75 μl of ddH2O <br /><br />
Note: Here listed the sequence of primers.<br /><br />
R-NPS-F 5'-TATAGCGGCCGCCTTAAGTAAGTAAGAGTATACG-3' <br /><br />
R-NPS-R 5'-CGGAGACTAGTCTGCAGATCACATAAGTAAAGTGATAATC-3' <br /><br />
G-SXA-F 5'-CTAGACTAGTTCTAGAGGCGGACTCACTATAGA-3' <br /><br />
G-SXA-R 5'-CCGACTTAAGGGATCCTATAAACGCAG-3'<br /><br />
2. Procedures on the thermocycler are listed below:<br /><br />
① 94˚C for 5 min<br /><br />
② 30 cycle<br /><br />
a. 94˚C for 1 min<br /><br />
b. 55˚C for 1 min<br /><br />
c. 72˚C for 1 min20sec<br /><br />
③ 4℃ for 7 hours </p><br />
</font><br />
<ul><br />
<li> Electrophorese the total system and observe the lane separation. </li><br />
</ul><br />
<p><strong>Figure</strong></p><br />
<p> <img src="https://static.igem.org/mediawiki/2012/0/07/1111.png" alt="" class="img_fl img_border" /> </p><br />
<p>(Figure 3: The lane on the figure are 2k fragments, it shows the GFP and RFP are ligated to the vectors which was isolated from the bacterial colonies.) </p><br />
<p><font face="Arial, Helvetica"><br><br />
<a name="Culture_the"></a><br />
</font></p><br />
<font face="Arial, Helvetica"><br />
</font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Cultivate the Bacteria</b></font></h3><br />
<font face="Arial, Helvetica"><p>According to the results of the PCR detection, we choose positive colonies and transfer them to 5ml LB liquid media ( 5μl of ampicillin has added) stored in 12.5ml centrifuge tubes. Put the centrifuge tubes in 37℃ gas bath overnight. </p><br />
<p><font face="Arial, Helvetica"><a name="Plasmid_DNA"></a> </font></p><br />
<font face="Arial, Helvetica"> </font> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Plasmid DNA Isolation</b></font></h3><br />
<p><font face="Arial, Helvetica">Use E.Z.N.A.TM Plasmid Mini I to isolate the constructed plasmid mutant-psb1a3-GR. </font></p><br />
<p><font face="Arial, Helvetica"><a name="Restriction_Enzyme" ></a> </font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Restriction Enzyme Digestion and Electrophoresis</b></font></h3><br />
<p>From the last step, we got the certain quantities of isolated plasmids. In this step, we do two restriction enzyme digestion reactions, one to prove that the plasmid is construct correctly ( mutant-psb1a3-GR ), one to get sticky ends preparing for the ligation.<br /><br />
<strong>Method</strong></p><br />
<ul><br />
<li><strong>Restriction Enzyme Digestion to prove that plasmid is constructed correctly</strong></li><br />
</ul><br />
<p align="left">a. Prepare the sample reaction as indicated below:<br /><br />
Total: 10μl<br /><br />
+ 1.0μl of Not I restriction enzyme,#ER0591<br /><br />
+ 1.0μl of Spe I restriction enzyme,#ER1251 <br /><br />
+ 2.0μl of Buffer Tango( 10X )<br /><br />
+ 1.5μl of plasmid mutant-psb1a3-GR<br /><br />
+ 14.5μl of ddH2O <br /><br />
b. Electrophorese the total system and observe the lane separation. </p><br />
<ul><br />
<li><strong>Restriction Enzyme Digestion to get sticky ends preparing for the ligation.</strong></li><br />
</ul><br />
<p align="left">a. Prepare the sample reaction as indicated below:<br /><br />
Total: 50μl<br /><br />
+ 5.0μl of Pst I restriction enzyme, #ER0611<br /><br />
+ 5.0μl of Xba I restriction enzyme, #ER0681 <br /><br />
+ 3.0μl of Buffer Tango( 10X )<br /><br />
+ 5.0μl of plasmid mutant-psb1a3-GR<br /><br />
+ 32.0μl of ddH2O <br /><br />
b. Electrophorese the total system and observe the lane separation.<br /><br />
c. Cut the gel of specific position and collect it in tubes that have measured weight. <br /><br />
d. Use DNA Gel Extraction Ki to purify the plasmid DNA mutant-psb1a3-GR(PX) .<br /><br />
Note: Mutant-psb1a3-GR(PX) means plasmid Mutant-psb1a3-GR digested by Pst I and Xba I.</p><br />
<p><strong>Figure</strong></p><br />
<p align="left"><strong><img src="https://static.igem.org/mediawiki/2012/d/d8/11111.png" alt="" class="img_fl img_border" /></strong></p><br />
<p>(Figure 4 : The double digestion of mutant-psb1a3 forms a linear DNA fragments and it runs slower than circle DNA fragments. This suggest that the double digestion of mutant-psb1a3 works in a high efficiency, and desired sticky ends are formed.1,3,5 are plasmids digested by restriction enzyme Pst I and Xba I from different colonies, 2,5,6 are pure plasmid mutant-psb1a3-GR, 7 is the plasmid mutant-psb1a3. )</p><br />
<p align="left">&nbsp;</p><br />
<p><font face="Arial, Helvetica"><a name="Ligation1" ></a> </font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Ligation</b></font></h3><br />
<p>Ligation is the process that target DNA gene is inserted into a plasmid. Both the vector and insert are prepared to have the sticky ends. These two kinds of DNA pieces are placed in a reaction tube and the proper DNA ligase, buffer, and cofactors are added for the reaction to take place. When done properly, the ligation will result in a successful combination of the insert and plasmid into one plasmid.Based on the digestion of mutant-psb1a3-GR, terminator DNA fragments,ligation can be done. We ligate mutant-psb1a3-GR vector and sticky terminator DNA fragments to construct an new plasmid mutant-psb1a3-GR-t.By detecting the quantities of GFP and RFP, terminator efficiency can be calculated. <br /><br />
1. Prepare the control reaction as indicated below:<br /><br />
Total: 10μl <br /><br />
+ 1.0μl of plasmid mutant-psb1a3 (NA) <br /><br />
+ 6.0μl of terminator<br /><br />
+ 2.0μl of T4 DNA Ligase , #EL0011 <br /><br />
+ 1.0μl of 10XT4 Ligase buffer<br /><br />
2. Put the tubes in 22℃ water bath, react for 8-12 hours. </p><br />
<p><font face="Arial, Helvetica"><a name="Bacteria" id="Culture_the"></a> </font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Bacteria transformation</b></font></h3><br />
<p>Transform the ligation products into the DH-5α competent cells, put the plate on 37℃ gas bath for 12-16 hours. </p><br />
<p><font face="Arial, Helvetica"><a name="Cultivate"></a> </font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Cultivate the Bacteria</b></font></h3><br />
<p>According to the results of the PCR detection, we choose positive colonies and transfer them to 5ml LB liquid media ( 5μl of ampicillin has added) stored in 12.5ml centrifuge tubes. Put the centrifuge tubes in 37℃ gas bath overnight. </p><br />
<p><font face="Arial, Helvetica"><a name="Flow" ></a> </font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Flow Cytometer Analysis</b></font></h3><br />
<p> 1. NaCl solution( 0.9% )<br /><br />
2. 75% Methanol<br /><br />
B. Procedures:<br /><br />
1. Transfer overnight suspension culture to 1.5ml centrifuge tubes and centrifuge at 12000RPM for 30s. Pour the supernatant and add overnight suspension culture and centrifuge again until enough bacteria has been collected. <br /><br />
2. Use NaCl solution(0.9%) to mix the bacteria and vibrate the centrifuge tubes until the bacteria distributed uniformly.<br /><br />
3. Put the centrifuge tubes into the Flow Cytometer and set parameters and run the program.</p><br />
<p>&nbsp;</p><br />
<p><font face="Arial, Helvetica"><a name="Fluorescence"></a> </font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Fluorescence Microscope</b></font></h3><br />
<p><strong>Method</strong><br /><br />
Add 10μl of former bacteria solution to micro slide and cover with coverslip.Then placed it on the Fluorescence Microscope with 488nm light activating and observe the GFP and RFP. </p><br />
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</html></div>M.B.ZHOUhttp://2012.igem.org/Team:SUSTC-Shenzhen-A/Biodesign_DownloadTeam:SUSTC-Shenzhen-A/Biodesign Download2012-10-26T19:23:52Z<p>M.B.ZHOU: </p>
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float: left;<br />
width: 180px;<br />
height: 196px; <br />
margin: 0 13px 12px 0; <br />
background: url(https://static.igem.org/mediawiki/igem.org/0/0d/Templatemo_portfolio_frame.jpg) no-repeat;<br />
}<br />
<br />
.showcase li a {<br />
display: block;<br />
margin: 20px;<br />
text-decoration: none;<br />
}<br />
<br />
.showcase li a img {<br />
margin-bottom: 5px;<br />
}<br />
<br />
.showcase li a span {<br />
margin-top: 5px;<br />
color: #5e5e5e;<br />
font-weight: normal;<br />
}<br />
/* end of content */<br />
<br />
/* footer */<br />
<br />
#templatemo_footer {<br />
clear: both;<br />
width: 1000px;<br />
padding: 10px 12px;<br />
margin: 0 auto;<br />
color: #252525;<br />
text-align: center;<br />
}<br />
<br />
#templatemo_footer a {<br />
font-weight: normal;<br />
color: #252525;<br />
text-decoration: underline;<br />
}<br />
<br />
/* end of footer */<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
</style><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
</head><br />
<body><br />
<br />
<a name="top"></a><br />
<a href="#top"><trytop></trytop></a><br />
<a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/SiteMap"><sitemap></sitemap></a><br />
<div id="templatemo_wrapper"><br />
<div id="temmplatmeo_header"><br />
<div id="site_title"><br />
<a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A" target="_parent"><br />
<img src="https://static.igem.org/mediawiki/2012/8/89/Sustclogo-1.png" width="270" height="74" alt="Logo" /><br />
</a><br />
</div><br />
<br />
<div id="templatemo_menu"><br />
<br />
<ul><br />
<li><a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A" class="current">Home</a></li><br />
<li><a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/Team" >Team</a></li><br />
<li><a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/Project">Project</a></li><br />
<li><a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/Notebook">Notebook</a></li><br />
<li ><a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/Human_practice">Human Practice</a></li><br />
</ul> <br />
<br />
</div> <!-- end of templatemo_menu --><br />
<br />
</div> <!-- end of templatemo_header --><br />
<br />
<div id="templatemo_banner"><br />
<br />
<div id="banner_left"><br />
<p class="title123">BioDesign & BioSearch</p><br />
</br><br />
<p class="title123">&nbsp;&nbsp;-- Synthetic Biology in Your Hand!</p><br />
</div><br />
<br />
<div id="banner_right"><span></span><br />
<br />
<div id="one" class="contentslider"><br />
<div class="cs_wrapper"><br />
<div class="cs_slider"><br />
<br />
<div class="cs_article"><br />
<img src="https://static.igem.org/mediawiki/2012/a/aa/School_view.jpg" width="517" height="215" alt="Seagulls"/><br />
</div><br />
<br />
<div class="cs_article"><br />
<img src="https://static.igem.org/mediawiki/igem.org/1/17/Members.JPG" width="517" height="215" alt="Seagulls"/><br />
</div><br />
<br />
<div class="cs_article"><br />
<a href="#" target="_parent"><br />
<img src="https://static.igem.org/mediawiki/2012/f/f3/Sustcproject1.jpg" width="517" height="215" alt="Seagulls"/><br />
</a></div><br />
<br />
</div><!-- End cs_slider --><br />
</div><!-- End cs_wrapper --><br />
</div><!-- End contentslider --><br />
<br />
<!-- Site JavaScript --><br />
<!-- 幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯 --><br />
<!-- 幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯 --><br />
<!-- 幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯 --><br />
<br />
<script type="text/javascript" src="http://taftproot.gotoip2.com/ld/js/jquery.easing.1.3.js"></script><br />
<script type="text/javascript" src="http://taftproot.gotoip2.com/ld/js/jquery.ennui.contentslider.js"></script><br />
<script type="text/javascript"><br />
$(function() {<br />
$('#one').ContentSlider({<br />
width : '535px',<br />
height : '233px',<br />
speed : 800,<br />
easing : 'easeInOutQuart'<br />
});<br />
});<br />
</script><br />
<script src="http://taftproot.gotoip2.com/ld/js/jquery.chili-2.2.js" type="text/javascript"></script><br />
<script src="http://taftproot.gotoip2.com/ld/js/chili/recipes.js" type="text/javascript"></script><br />
<br />
<br />
</div> <!-- end of slider --> <br />
<!-- 幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯 --><br />
<!-- 幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯 --><br />
<!-- 幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯幻灯 --><br />
<br />
<br />
</div> <!-- end of templatemo_banner --><br />
<br />
<br />
</div> <!-- end of templatemo_wrapper --><br />
<br />
<!-- Thanks to http://www.qianduan.net/fresh-free-html-templates-2010.html--><br />
</body><br />
<br />
</html></div>M.B.ZHOUhttp://2012.igem.org/Template:SUSTC_ATemplate:SUSTC A2012-10-26T19:00:26Z<p>M.B.ZHOU: </p>
<hr />
<div><html xmlns="http://www.w3.org/1999/xhtml"><br />
<head><br />
<meta http-equiv="Content-Type" content="text/html; charset=utf-8" /><br />
<title></title><br />
<style type="text/css"><br />
trytop<br />
{<br />
position:fixed; bottom:0px; right:0px;<br />
background:url("https://static.igem.org/mediawiki/2012/a/a8/SustcTop.png") no-repeat;<br />
background-position: 0px 0px;<br />
display: inline;<br />
float: left;<br />
height: 60px;<br />
width:60px;<br />
<br />
margin-bottom: 0;<br />
}<br />
<br />
sitemap<br />
{<br />
position:fixed; bottom:0px; left:0px;<br />
background:url("https://static.igem.org/mediawiki/2012/e/e7/SUSTCSitemap.png") no-repeat;<br />
background-position: 0px 0px;<br />
display: inline;<br />
float: left;<br />
height: 60px;<br />
width:60px;<br />
<br />
margin-bottom: 0;<br />
}<br />
<br />
<br />
<!--我们需要先把igem原网站的一些东西删掉--><br />
#globalWrapper {width: 100%; }<br />
#top-section {width: 100%; height:100%; border:none;}<br />
#p-logo {display:none;}<br />
#search-controls {display:none;}<br />
.printfooter {display:none;}<br />
#footer-box {border:none;}<br />
.firstHeading {display:none;}<br />
#content { border:none; width:1024px; }<br />
#bodyContent {border:none; }<br />
<br />
<br />
#catlinks {display:none; }<br />
<br />
#footer-box {display:none; }<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<!--删除完毕--><br />
<br />
<br />
<br />
/*<br />
* Color, Border, and Button Rules<br />
*/<br />
.contentslider {<br />
padding: 0; /* This acts as a border for the content slider */<br />
background: none; /* This is the color of said border */<br />
overflow:hidden;<br />
} <br />
.cs_wrapper, .cs_article {<br />
/* background-color: none; Background color for the entries */<br />
}<br />
.cs_leftBtn, .cs_rightBtn {<br />
margin-top: 50px;<br />
width:52px; /* Should be as wide as the button graphic being used */<br />
/* background: none; This will probably match the contentslider bg color */<br />
}<br />
<br />
/*<br />
* Article styles (font, color, etc.)<br />
*<br />
* If textResize is set to TRUE, sizing shouldn't need to be touched. However,<br />
* depending on the sizes you have defined, additional tweaking may be<br />
* required in order to get the text to display properly.<br />
*/<br />
<br />
/*<br />
******************************************************************************<br />
* These styles may be affected by the plugin, so avoid changing them if <br />
* it's not absolutely necessary.<br />
******************************************************************************<br />
*/<br />
.contentslider {<br />
position:relative;<br />
display:block;<br />
width: 535px;<br />
height: 233px;<br />
margin:0 auto;<br />
overflow:hidden;<br />
}<br />
.cs_wrapper {<br />
position:relative;<br />
display:block;<br />
width:100%;<br />
height:100%;<br />
margin:0;<br />
padding:0;<br />
overflow:hidden;<br />
}<br />
.cs_slider {<br />
position:absolute;<br />
width:10000px;<br />
height:100%;<br />
margin:0;<br />
padding:0;<br />
overflow:hidden;<br />
}<br />
.cs_article {<br />
float:left;<br />
position:relative;<br />
top:0;<br />
left:0;<br />
display:block;<br />
width: 535px;<br />
height: 233px;<br />
margin:0 auto;<br />
padding: 0;<br />
/* background-color: none; */<br />
overflow:hidden;<br />
}<br />
<br />
.cs_article img {<br />
float: left;<br />
}<br />
<br />
.cs_leftBtn, .cs_rightBtn {<br />
position:absolute;<br />
top: 0px;<br />
height: 233px;<br />
padding:0;<br />
z-index:10000;<br />
}<br />
.cs_leftBtn {<br />
left:0;<br />
outline:0;<br />
}<br />
.cs_rightBtn {<br />
right:0;<br />
outline:0;<br />
}<br />
.cs_leftBtn img, .cs_rightBtn img {<br />
border:0;<br />
position:relative;<br />
top: 10px;<br />
margin:0;<br />
}<br />
<br />
<br />
<br />
<br />
/* Div IDs */ /***********/ body, #content,#table {<br />
background: transparent;<br />
}<br />
<br />
<br />
body {<br />
margin: 0;<br />
padding: 0;<br />
line-height: 1.5em;<br />
font-family: Tahoma, Geneva, sans-serif;<br />
font-size: 10px;<br />
color: #6f6f6f;<br />
background: #2ac5c0 url(https://static.igem.org/mediawiki/2012/e/e8/Templatemo_body_top2.jpg) repeat-x;<br />
}<br />
<br />
a:link, a:visited { color: #17aba6; text-decoration: underline; font-weight: bold; } <br />
a:active, a:hover { color: #CC9900; text-decoration: none; }<br />
<br />
p { margin: 0px; padding: 0px; }<br />
<br />
img { margin: 0px; padding: 0px; border: none; }<br />
<br />
.cleaner { clear: both; width: 100%; height: 0px; font-size: 0px; }<br />
<br />
.cleaner_h30 { clear: both; width:100%; height: 30px; }<br />
.cleaner_h40 { clear: both; width:100%; height: 40px; }<br />
<br />
.margin_r10 { margin-right: 10px; }<br />
<br />
.float_l { float: left; }<br />
.float_r { float: right; }<br />
<br />
<br />
#templatemo_wrapper {<br />
width: 1024px;<br />
margin: 0 auto;<br />
background-color:transparent;<br />
}<br />
<br />
#temmplatmeo_header {<br />
height: 96px;<br />
background: url(http://taftproot.gotoip2.com/ld/images/templatemo_menu.jpg) no-repeat bottom;<br />
}<br />
<br />
/* site title */<br />
<br />
#temmplatmeo_header #site_title {<br />
float: left;<br />
width: 200px;<br />
padding: 10px 0 0 50px;<br />
background-color:transparent;<br />
}<br />
<br />
#site_title a {<br />
margin: 0px;<br />
padding: 0px;<br />
font-size: 30px;<br />
color: #ffffff;<br />
font-weight: bold;<br />
text-decoration: none;<br />
background-color:transparent;<br />
}<br />
<br />
#site_title h1 a:hover {<br />
font-weight: bold; <br />
text-decoration: none;<br />
background-color:transparent;<br />
}<br />
<br />
/* end of site title */<br />
<br />
/* menu */<br />
<br />
#temmplatmeo_header #templatemo_menu {<br />
float: right;<br />
padding-top: 53px;<br />
height: 43px;<br />
margin-right: 70px<br />
}<br />
<br />
#templatemo_menu ul {<br />
margin: 0;<br />
padding: 0;<br />
list-style: none;<br />
}<br />
<br />
#templatemo_menu ul li {<br />
padding: 0;<br />
margin: 0;<br />
display: inline;<br />
}<br />
<br />
#templatemo_menu ul li a {<br />
position: relative;<br />
float: left;<br />
display: block;<br />
height: 33px;<br />
width: 123px;<br />
margin: 0 0 0 -20px;<br />
padding: 10px 0 0 20px;<br />
font-family: "Times New Roman", Times, serif;<br />
text-align: center;<br />
font-size: 16px;<br />
text-decoration: none;<br />
color: #fff; <br />
font-weight: normal;<br />
outline: none;<br />
background:url(https://static.igem.org/mediawiki/igem.org/e/e8/Templatemo_menu_button.png) no-repeat;<br />
}<br />
<br />
#templatemo_menu li a:hover, #templatemo_menu li .current {<br />
z-index: 200;<br />
background:url(https://static.igem.org/mediawiki/2012/8/82/SUSTC_SHENZHEN_A_temp_menu_button_hover.png);<br />
}<br />
<br />
/* end of menu */<br />
<br />
/* banner */<br />
<br />
#templatemo_banner {<br />
width: 954px;<br />
height: 236px;<br />
padding: 50px 35px;<br />
background: url(https://static.igem.org/mediawiki/igem.org/1/17/Templatemo_banner.png) no-repeat;<br />
}<br />
<br />
#banner_left {<br />
float: left;<br />
width: 365px;<br />
color: #fff;<br />
text-align: justify;<br />
}<br />
<br />
#banner_left h2 {<br />
font-family: "Comic Sans MS", cursive;<br />
font-size: 30px;<br />
font-weight: normal;<br />
margin-top: 0;<br />
<br />
}<br />
#banner_left h3 {<br />
font-family: "Comic Sans MS", cursive;<br />
font-size: 32px;<br />
<br />
}<br />
<br />
#banner_left h4 {<br />
font-family: "Comic Sans MS", cursive;<br />
font-size: 28px;<br />
<br />
}<br />
<br />
#banner_left a {<br />
color: #1cc9c4;<br />
}<br />
<br />
#banner_right {<br />
position: relative;<br />
float: right;<br />
width: 517px;<br />
height: 215px;<br />
padding: 9px;<br />
margin-right: 30px;<br />
overflow: hidden;<br />
}<br />
<br />
#banner_right span {<br />
position: absolute;<br />
width: 535px;<br />
height: 233px;<br />
top: 0;<br />
left: 0;<br />
z-index: 800;<br />
background: url(https://static.igem.org/mediawiki/igem.org/6/62/Templatemo_banner_image_frame.png) no-repeat;<br />
}<br />
/* end of banner */<br />
<br />
/* content */<br />
<br />
#templatemo_content {<br />
<br />
clear: both;<br />
width: 900px;<br />
padding: 35px 62px 15px 62px;<br />
background: url(https://static.igem.org/mediawiki/igem.org/8/89/Templatemo_content_bg.png) repeat-y center;<br />
}<br />
<br />
#content_bottom {<br />
width: 1024px;<br />
height: 6px;<br />
background: url(images/templatemo_content_bottom.png) no-repeat;<br />
}<br />
<br />
.float_l_img {<br />
float: left;<br />
margin: 3px 15px 5px 0;<br />
}<br />
<br />
#templatemo_content h2 {<br />
color: #9ea0a1;<br />
font-weight: normal;<br />
font-size: 25px;<br />
margin: 0 0 25px 0;<br />
}<br />
<br />
.title123 {<br />
font-family: "Comic Sans MS", cursive;<br />
font-size: xx-large;<br />
color: #ffffff;<br />
}<br />
<br />
#templatemo_content p {<br />
margin-bottom: 6px;<br />
text-align: justify;<br />
}<br />
<br />
#templatemo_content strong {<br />
color: #17aba6;<br />
font-weight: bold; <br />
}<br />
<br />
#templatemo_content .services_list {<br />
margin: 20px 150px 0 0;<br />
padding: 0;<br />
list-style: none;<br />
}<br />
<br />
#templatemo_content .services_list li {<br />
margin: 0 0 5px 0;<br />
padding: 4px 0 5px 15px;<br />
border-bottom: 1px dashed #c7c7c7;<br />
background: url(https://static.igem.org/mediawiki/igem.org/a/ac/List_icon.png) no-repeat center left;<br />
}<br />
<br />
.section_w900 {<br />
clear: both;<br />
width: 900px;<br />
}<br />
<br />
.section_w580 {<br />
width: 580px;<br />
}<br />
<br />
.section_w280 {<br />
float: right;<br />
width: 280px;<br />
}<br />
<br />
.services_section {<br />
clear: both;<br />
padding: 20px;<br />
background: url(https://static.igem.org/mediawiki/igem.org/3/3e/Templatemo_services.jpg) no-repeat top center;<br />
}<br />
<br />
.testimonial {<br />
<br />
height: 160px;<br />
padding: 60px 20px 20px 70px;<br />
margin-bottom: 30px;<br />
overflow: hidden;<br />
background:url(https://static.igem.org/mediawiki/igem.org/8/81/Templatemo_testimonial.png) no-repeat;<br />
text-align: justify;<br />
}<br />
<br />
.twitter {<br />
width: 280px;<br />
height: 180px;<br />
overflow: hidden;<br />
background: url(https://static.igem.org/mediawiki/igem.org/a/a4/Templatemo_twitter.jpg) no-repeat center;<br />
text-align: justify;<br />
}<br />
<br />
.twitter ul {<br />
margin: 0;<br />
padding: 70px 30px 30px 30px;<br />
list-style: none;<br />
}<br />
<br />
.twitter ul li {<br />
margin: 0 0 10px 0;<br />
padding: 0;<br />
}<br />
<br />
.twitter ul li span {<br />
clear: both;<br />
display: block;<br />
font-style: italic;<br />
color: #26aba7;<br />
}<br />
<br />
.showcase {<br />
margin: 0;<br />
padding: 0;<br />
list-style: none;<br />
}<br />
<br />
.showcase li {<br />
margin: 0;<br />
padding: 0;<br />
display: block;<br />
float: left;<br />
width: 180px;<br />
height: 196px; <br />
margin: 0 13px 12px 0; <br />
background: url(https://static.igem.org/mediawiki/igem.org/0/0d/Templatemo_portfolio_frame.jpg) no-repeat;<br />
}<br />
<br />
.showcase li a {<br />
display: block;<br />
margin: 20px;<br />
text-decoration: none;<br />
}<br />
<br />
.showcase li a img {<br />
margin-bottom: 5px;<br />
}<br />
<br />
.showcase li a span {<br />
margin-top: 5px;<br />
color: #5e5e5e;<br />
font-weight: normal;<br />
}<br />
/* end of content */<br />
<br />
/* footer */<br />
<br />
#templatemo_footer {<br />
clear: both;<br />
width: 1000px;<br />
padding: 10px 12px;<br />
margin: 0 auto;<br />
color: #252525;<br />
text-align: center;<br />
}<br />
<br />
#templatemo_footer a {<br />
font-weight: normal;<br />
color: #252525;<br />
text-decoration: underline;<br />
}<br />
<br />
/* end of footer */<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
</style><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
</head><br />
<body><br />
<br />
<a name="top"></a><br />
<a href="#top"><trytop></trytop></a><br />
<a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/SiteMap"><sitemap></sitemap></a><br />
<div id="templatemo_wrapper"><br />
<div id="temmplatmeo_header"><br />
<div id="site_title"><br />
<a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A" target="_parent"><br />
<img src="https://static.igem.org/mediawiki/2012/8/89/Sustclogo-1.png" width="270" height="74" alt="Logo" /><br />
</a><br />
</div><br />
<br />
<div id="templatemo_menu"><br />
<br />
<ul><br />
<li><a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A" class="current">Home</a></li><br />
<li><a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/Team" >Team</a></li><br />
<li><a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/Project">Project</a></li><br />
<li><a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/Notebook">Notebook</a></li><br />
<li ><a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/Human_practice">Human Practice</a></li><br />
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<p class="title123">BioDesign&BioSearch</p><br />
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<p class="title123">&nbsp;&nbsp;--Make Synthetic Biology in Your Hand!</p><br />
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</html></div>M.B.ZHOUhttp://2012.igem.org/Team:SUSTC-Shenzhen-ATeam:SUSTC-Shenzhen-A2012-10-26T18:58:11Z<p>M.B.ZHOU: </p>
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<p class="title1">BioDesign Abstract</p><br />
<img src="https://static.igem.org/mediawiki/2012/c/c9/Devidingline_side.jpg"><br />
<p>&nbsp;</p><br />
<br />
<p>&nbsp;&nbsp;You can draw a biological circuit with your freehand on iPhone with BioDesign!</p><br />
<p>&nbsp;&nbsp;Biodesign is a graphics editing application specific to synthetic biology for iPhone. It is based on Tinkercell and provides hundreds of biobrick icons as well as a great many fancy functions including dragging graphics, drawing curves and ect. With BioSearch, users are capable of drawing and sharing biochemical reaction schematics whenever and wherever they want. Biodesign will be available online very soon!</p><br />
<p>&nbsp;</p><br />
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<img src="https://static.igem.org/mediawiki/2012/c/c9/Devidingline_side.jpg"><br />
<p>&nbsp;</p><br />
<p>&nbsp;&nbsp;The era of Partsregistry on mobile phone has arrived! We developed an iPhone App for partsregistry website, <a href="http://partsregistry.org/Main_Page">http://partsregistry.org</a>. With Biosearch on your iPhone, you can now check BioBricks and partsregistry in the seminar room; You can design your genetic circuits when you are waiting for a bus!</p><br />
<p>&nbsp;&nbsp;Biosearch is fully interacting with Partsregistry and has all parts information of Partsregistry database with enhanced user-friendly interface.</p><br />
<p>&nbsp;</p><br />
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[[file:footbar.jpg|center]]</div>M.B.ZHOUhttp://2012.igem.org/Team:SUSTC-Shenzhen-A/Biodesign_UpdateTeam:SUSTC-Shenzhen-A/Biodesign Update2012-10-26T18:50:57Z<p>M.B.ZHOU: </p>
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<h1 class="title">Future Plan</h1><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title1">Key Features in BioDesign V2.0</p><br />
<p>&nbsp;&nbsp;BioSearch is just a 2-month old software developed entirely by a group of passionate undergraduate novices from SUSTC who dare to challenge the unchallengeable. During the past 2 months, we are making every effort to optimize it incessantly. BioSearch still has a long way to go. With careful analysis and prudent evaluation of our application, here we make a list of improvements waiting to be implemented in next version.</p><br />
<p>&nbsp;&nbsp;</p><br />
<strong>iPad version & Android Version</strong><br />
<p>&nbsp;&nbsp;To better satisfy the need of increasing iPad/Android user.</p><br />
<p>&nbsp;&nbsp;</p><br />
<strong>Merge BioDesign with BioSearch</strong><br />
<p>&nbsp;&nbsp;We expect to import data from BioSearch to BioDesign to do biochemical reaction simulation. Then BioSearch will be even stronger and comprehensive.</p><br />
<p>&nbsp;&nbsp;</p><br />
<strong>Simulation & Calculation</strong><br />
<p>&nbsp;&nbsp;What if BioDesign is more than a graphic editing program? Many potential users expected BioDesign to possess simulation function and calculation function. Perhaps we need first to find out the algorithm of simulation and then to take the advantage of cloud calculation via the Internet to make our App more comprehensive. </p><br />
<p>&nbsp;&nbsp;</p><br />
<strong>Optimization of User Interface & Graphics Editing Module</strong><br />
<p>&nbsp;&nbsp;Currently, BioDesign has achieved all the essential graphic editing functions, which already satisfied the primary requirements of potential users and researchers. However, the layout of the user interface can be improved to be more reasonable and beautiful if time permitted.</p> <br />
<p>&nbsp;&nbsp;</p><br />
<strong>Redo/Undo/Cut/Copy</strong><br />
<p>&nbsp;&nbsp;o far, you can use BioDesign to add/delete/rotate/connect figures. However, to better provide convenience to users, we’d like to develop redo/undo/cut/copy functions to cater for users’ habit. These problems are still challenging because these involved much more complex data storage and data extraction. If these problems are not proper-handled, RAM are vulnerable to run out of space. Therefore, we are still trying to overcome those difficulties and to update the App as soon as possible.</p><br />
<p>&nbsp;&nbsp;</p><br />
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[[file:footbar.jpg|center]]</div>M.B.ZHOUhttp://2012.igem.org/Team:SUSTC-Shenzhen-A/week13Team:SUSTC-Shenzhen-A/week132012-10-26T18:29:53Z<p>M.B.ZHOU: </p>
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<br />
<table width="899" border="0" cellspacing="10px" cellpadding="10px" align="center" height="870"><br />
<tr><br />
<td valign="top"><br />
<div id="talkbubble_b"><br />
<h1 class="title">Week13~Week18</h1><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;</p><br />
<ul><br />
<br />
<li>Week13</li><br />
</ul><br />
<br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;Asia Jamboree in HKUST!</p><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;We met many teams from China, Japan, Korea, India and Indoneisa. We presented a high quality presentation. Yet we received many good advices from the judges. And we showed our poster and made many friends! But it is a little upset that we only got a silver medal. We think that we deserved a gold medal. Because we think we fulfilled every requirements of a gold medal. </p><br />
<br/><br/><br />
<ul><br />
<li>Week 14, 15</li><br />
</ul><br />
<br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;First two weeks from Hong Kong. We were a little upset since we were not invited to MIT. So we did nothing these two weeks. </p><br />
<p>&nbsp;&nbsp;</p><br />
<br />
<br />
<ul><br />
<li>Week 16</li><br />
</ul><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;Good news! We were invited to MIT, too! We came back to our second project---BioDesign. Xiao Tong and Zili Fan were in charge of the drawing part. They spent virtually all the spare time on this project. Sometimes they even skipped classes to write code. Mubing Zhou was in charge of the rest part other than the drawing part. He was also very hardworking. Deng Pan and Yujun Zhao were in charge of updating BioSearch. While Qijia Cheng, Chenchen Lyu and Junqiu Zhang were busy preparing the presentation. Yidan Pan and Xin Yang were in charge of the wiki writing. And Jingyao Guo and Yiqi Jiang were in charge of name card making and poster redesigning.</p><br />
<p>&nbsp;&nbsp;</p><br />
<br />
<ul><br />
<li>Week 17</li><br />
</ul><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;Since we had had some experience in writing codes, we didn't discuss too much when developing BioDesign. This week. We kept working hard. Xiao Tong, Zili Fan and Mubing Zhou already finished BioDesign. But there was a huge task remaining--debugging. Hundreds of bugs were hidden between lines. Sometimes, they spent half a day just to fix a tiny problem. Other guys were still doing their jobs. Jingyao Guo and Yiqi Jiang printed our name cards and new poster. Deng Pan and Yujun Zhao fixed some bugs in BioSearch.</p><br />
<p>&nbsp;&nbsp;</p><br />
<br />
<ul><br />
<li>Week 18</li><br />
</ul><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;World Jamboree in MIT! </p><br />
<br />
</div><br />
<br />
</td><br />
<br />
<br />
<br />
<br />
<td><div id="talkbubble1"><div class="sidebar_box"><div class="sidebar_box_top"></div><br />
<div class="sidebar_box_content"> <br />
<p class="title1">Notebook</p><br />
<img src="https://static.igem.org/mediawiki/2012/c/c9/Devidingline_side.jpg"><br />
<p><a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/Notebook"><strong>Overview</strong></a></p><br />
<p><strong>Preparation</strong></p><br />
<p><a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/JC"><strong>Journal Club</strong></a></p><br />
<p><a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/FP"><strong>Final Project</strong></a></p><br />
<p><a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/week1"><strong>Xcode Tutorial(week 1)</strong></a></p><br />
<p><strong>Biosearch section1(week2,3)</strong></p><br />
<p>&nbsp;&nbsp;<a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/week2">week 2</a></p><br />
<p>&nbsp;&nbsp;<a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/week3">week 3</a></p><br />
<p><strong>Biosearch section2(week4,5)</strong></p><br />
<p><a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/week4&5"><strong>week4~5</strong></a></p><br />
<p><strong>Tinker Cell(week,6,7)</strong></p><br />
<p>&nbsp;&nbsp;<a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/week6">week 6</a></p><br />
<p>&nbsp;&nbsp;<a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/week7">week 7</a></p><br />
<p><strong>Comprehensive work</strong></p><br />
<p>&nbsp;&nbsp;<a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/week8">week 8</a></p><br />
<p>&nbsp;&nbsp;<a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/week9">week 9~12</a></p><br />
<p>&nbsp;&nbsp;<a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/week13">week 13~18</a></p><br />
<br />
<div class="sidebar_box_bottom"></div> <br />
</div><br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
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[[file:footbar.jpg|center]]</div>M.B.ZHOUhttp://2012.igem.org/Team:SUSTC-Shenzhen-A/week13Team:SUSTC-Shenzhen-A/week132012-10-26T18:28:29Z<p>M.B.ZHOU: </p>
<hr />
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</head><br />
<body ><br />
<br />
<table width="899" border="0" cellspacing="10px" cellpadding="10px" align="center" height="870"><br />
<tr><br />
<td valign="top"><br />
<div id="talkbubble_b"><br />
<h1 class="title">Week13~Week18</h1><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;</p><br />
<ul><br />
<br />
<li>Week13</li><br />
</ul><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;</p><br />
<br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;Asia Jamboree in HKUST!</p><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;We met many teams from China, Japan, Korea, India and Indoneisa. We presented a high quality presentation. Yet we received many good advices from the judges. And we showed our poster and made many friends! But it is a little upset that we only got a silver medal. We think that we deserved a gold medal. Because we think we fulfilled every requirements of a gold medal. </p><br />
<br/><br/><br />
<ul><br />
<li>Week 14, 15</li><br />
</ul><br />
<br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;First two weeks from Hong Kong. We were a little upset since we were not invited to MIT. So we did nothing these two weeks. </p><br />
<p>&nbsp;&nbsp;</p><br />
<br />
<br />
<ul><br />
<li>Week 16</li><br />
</ul><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;Good news! We were invited to MIT, too! We came back to our second project---BioDesign. Xiao Tong and Zili Fan were in charge of the drawing part. They spent virtually all the spare time on this project. Sometimes they even skipped classes to write code. Mubing Zhou was in charge of the rest part other than the drawing part. He was also very hardworking. Deng Pan and Yujun Zhao were in charge of updating BioSearch. While Qijia Cheng, Chenchen Lyu and Junqiu Zhang were busy preparing the presentation. Yidan Pan and Xin Yang were in charge of the wiki writing. And Jingyao Guo and Yiqi Jiang were in charge of name card making and poster redesigning.</p><br />
<br />
<ul><br />
<li>Week 17</li><br />
</ul><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;Since we had had some experience in writing codes, we didn't discuss too much when developing BioDesign. This week. We kept working hard. Xiao Tong, Zili Fan and Mubing Zhou already finished BioDesign. But there was a huge task remaining--debugging. Hundreds of bugs were hidden between lines. Sometimes, they spent half a day just to fix a tiny problem. Other guys were still doing their jobs. Jingyao Guo and Yiqi Jiang printed our name cards and new poster. Deng Pan and Yujun Zhao fixed some bugs in BioSearch.</p><br />
<br />
<ul><br />
<li>Week 18</li><br />
</ul><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;World Jamboree in MIT! </p><br />
<br />
</div><br />
<br />
</td><br />
<br />
<br />
<br />
<br />
<td><div id="talkbubble1"><div class="sidebar_box"><div class="sidebar_box_top"></div><br />
<div class="sidebar_box_content"> <br />
<p class="title1">Notebook</p><br />
<img src="https://static.igem.org/mediawiki/2012/c/c9/Devidingline_side.jpg"><br />
<p><a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/Notebook"><strong>Overview</strong></a></p><br />
<p><strong>Preparation</strong></p><br />
<p><a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/JC"><strong>Journal Club</strong></a></p><br />
<p><a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/FP"><strong>Final Project</strong></a></p><br />
<p><a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/week1"><strong>Xcode Tutorial(week 1)</strong></a></p><br />
<p><strong>Biosearch section1(week2,3)</strong></p><br />
<p>&nbsp;&nbsp;<a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/week2">week 2</a></p><br />
<p>&nbsp;&nbsp;<a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/week3">week 3</a></p><br />
<p><strong>Biosearch section2(week4,5)</strong></p><br />
<p><a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/week4&5"><strong>week4~5</strong></a></p><br />
<p><strong>Tinker Cell(week,6,7)</strong></p><br />
<p>&nbsp;&nbsp;<a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/week6">week 6</a></p><br />
<p>&nbsp;&nbsp;<a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/week7">week 7</a></p><br />
<p><strong>Comprehensive work</strong></p><br />
<p>&nbsp;&nbsp;<a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/week8">week 8</a></p><br />
<p>&nbsp;&nbsp;<a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/week9">week 9~12</a></p><br />
<p>&nbsp;&nbsp;<a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/week13">week 13~18</a></p><br />
<br />
<div class="sidebar_box_bottom"></div> <br />
</div><br />
</td><br />
</tr><br />
</table><br />
<br />
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<br />
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</html><br />
[[file:footbar.jpg|center]]</div>M.B.ZHOUhttp://2012.igem.org/Team:SUSTC-Shenzhen-B/project.overviewTeam:SUSTC-Shenzhen-B/project.overview2012-10-26T18:15:12Z<p>M.B.ZHOU: </p>
<hr />
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<br />
/* prettyPhoto.css */<br />
div.pp_default .pp_top,div.pp_default .pp_top .pp_middle,div.pp_default .pp_top .pp_left,div.pp_default .pp_top .pp_right,div.pp_default .pp_bottom,div.pp_default .pp_bottom .pp_left,div.pp_default .pp_bottom .pp_middle,div.pp_default .pp_bottom .pp_right{height:13px}div.pp_default .pp_top .pp_left{background:url(../images/default/sprite.png) -78px -93px no-repeat}div.pp_default .pp_top .pp_middle{background:url(../images/default/sprite_x.png) top left repeat-x}div.pp_default .pp_top .pp_right{background:url(../images/default/sprite.png) -112px -93px no-repeat}div.pp_default .pp_content .ppt{color:#f8f8f8}div.pp_default .pp_content_container .pp_left{background:url(../images/default/sprite_y.png) -7px 0 repeat-y;padding-left:13px}div.pp_default .pp_content_container .pp_right{background:url(../images/default/sprite_y.png) top right repeat-y;padding-right:13px}div.pp_default .pp_next:hover{background:url(../images/default/sprite_next.png) center right no-repeat;cursor:pointer}div.pp_default .pp_previous:hover{background:url(../images/default/sprite_prev.png) center left no-repeat;cursor:pointer}div.pp_default .pp_expand{background:url(../images/default/sprite.png) 0 -29px no-repeat;cursor:pointer;height:28px;width:28px}div.pp_default .pp_expand:hover{background:url(../images/default/sprite.png) 0 -56px no-repeat;cursor:pointer}div.pp_default .pp_contract{background:url(../images/default/sprite.png) 0 -84px no-repeat;cursor:pointer;height:28px;width:28px}div.pp_default .pp_contract:hover{background:url(../images/default/sprite.png) 0 -113px no-repeat;cursor:pointer}div.pp_default .pp_close{background:url(../images/default/sprite.png) 2px 1px no-repeat;cursor:pointer;height:30px;width:30px}div.pp_default .pp_gallery ul li a{background:url(../images/default/default_thumb.png) center center #f8f8f8;border:1px solid #aaa}div.pp_default .pp_social{margin-top:7px}div.pp_default .pp_gallery a.pp_arrow_previous,div.pp_default .pp_gallery a.pp_arrow_next{left:auto;position:static}div.pp_default .pp_nav .pp_play,div.pp_default .pp_nav .pp_pause{background:url(../images/default/sprite.png) -51px 1px no-repeat;height:30px;width:30px}div.pp_default .pp_nav .pp_pause{background-position:-51px -29px}div.pp_default a.pp_arrow_previous,div.pp_default a.pp_arrow_next{background:url(../images/default/sprite.png) -31px -3px no-repeat;height:20px;margin:4px 0 0;width:20px}div.pp_default a.pp_arrow_next{background-position:-82px -3px;left:52px}div.pp_default .pp_content_container .pp_details{margin-top:5px}div.pp_default .pp_nav{clear:none;height:30px;position:relative;width:110px}div.pp_default .pp_nav .currentTextHolder{color:#999;font-family:Georgia;font-size:11px;font-style:italic;left:75px;line-height:25px;margin:0;padding:0 0 0 10px;position:absolute;top:2px}div.pp_default .pp_close:hover,div.pp_default .pp_nav .pp_play:hover,div.pp_default .pp_nav .pp_pause:hover,div.pp_default .pp_arrow_next:hover,div.pp_default .pp_arrow_previous:hover{opacity:.7}div.pp_default .pp_description{font-size:11px;font-weight:700;line-height:14px;margin:5px 50px 5px 0}div.pp_default .pp_bottom .pp_left{background:url(../images/default/sprite.png) -78px -127px no-repeat}div.pp_default .pp_bottom .pp_middle{background:url(../images/default/sprite_x.png) bottom left repeat-x}div.pp_default .pp_bottom .pp_right{background:url(../images/default/sprite.png) -112px -127px no-repeat}div.pp_default .pp_loaderIcon{background:url(../images/default/loader.gif) center center no-repeat}div.light_rounded .pp_top .pp_left{background:url(../images/light_rounded/sprite.png) -88px -53px no-repeat}div.light_rounded .pp_top .pp_right{background:url(../images/light_rounded/sprite.png) -110px -53px no-repeat}div.light_rounded .pp_next:hover{background:url(../images/light_rounded/btnNext.png) center right no-repeat;cursor:pointer}div.light_rounded .pp_previous:hover{background:url(../images/light_rounded/btnPrevious.png) center left no-repeat;cursor:pointer}div.light_rounded .pp_expand{background:url(../images/light_rounded/sprite.png) -31px -26px no-repeat;cursor:pointer}div.light_rounded .pp_expand:hover{background:url(../images/light_rounded/sprite.png) -31px -47px no-repeat;cursor:pointer}div.light_rounded .pp_contract{background:url(../images/light_rounded/sprite.png) 0 -26px no-repeat;cursor:pointer}div.light_rounded .pp_contract:hover{background:url(../images/light_rounded/sprite.png) 0 -47px no-repeat;cursor:pointer}div.light_rounded .pp_close{background:url(../images/light_rounded/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:75px}div.light_rounded .pp_nav .pp_play{background:url(../images/light_rounded/sprite.png) -1px -100px no-repeat;height:15px;width:14px}div.light_rounded .pp_nav .pp_pause{background:url(../images/light_rounded/sprite.png) -24px -100px no-repeat;height:15px;width:14px}div.light_rounded .pp_arrow_previous{background:url(../images/light_rounded/sprite.png) 0 -71px no-repeat}div.light_rounded .pp_arrow_next{background:url(../images/light_rounded/sprite.png) -22px -71px no-repeat}div.light_rounded .pp_bottom .pp_left{background:url(../images/light_rounded/sprite.png) -88px -80px no-repeat}div.light_rounded .pp_bottom .pp_right{background:url(../images/light_rounded/sprite.png) -110px -80px no-repeat}div.dark_rounded .pp_top .pp_left{background:url(../images/dark_rounded/sprite.png) -88px -53px no-repeat}div.dark_rounded .pp_top .pp_right{background:url(../images/dark_rounded/sprite.png) -110px -53px no-repeat}div.dark_rounded .pp_content_container .pp_left{background:url(../images/dark_rounded/contentPattern.png) top left repeat-y}div.dark_rounded .pp_content_container .pp_right{background:url(../images/dark_rounded/contentPattern.png) top right repeat-y}div.dark_rounded .pp_next:hover{background:url(../images/dark_rounded/btnNext.png) center right no-repeat;cursor:pointer}div.dark_rounded .pp_previous:hover{background:url(../images/dark_rounded/btnPrevious.png) center left no-repeat;cursor:pointer}div.dark_rounded .pp_expand{background:url(../images/dark_rounded/sprite.png) -31px -26px no-repeat;cursor:pointer}div.dark_rounded .pp_expand:hover{background:url(../images/dark_rounded/sprite.png) -31px -47px no-repeat;cursor:pointer}div.dark_rounded .pp_contract{background:url(../images/dark_rounded/sprite.png) 0 -26px no-repeat;cursor:pointer}div.dark_rounded .pp_contract:hover{background:url(../images/dark_rounded/sprite.png) 0 -47px no-repeat;cursor:pointer}div.dark_rounded .pp_close{background:url(../images/dark_rounded/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:75px}div.dark_rounded .pp_description{color:#fff;margin-right:85px}div.dark_rounded .pp_nav .pp_play{background:url(../images/dark_rounded/sprite.png) -1px -100px no-repeat;height:15px;width:14px}div.dark_rounded .pp_nav .pp_pause{background:url(../images/dark_rounded/sprite.png) -24px -100px no-repeat;height:15px;width:14px}div.dark_rounded .pp_arrow_previous{background:url(../images/dark_rounded/sprite.png) 0 -71px no-repeat}div.dark_rounded .pp_arrow_next{background:url(../images/dark_rounded/sprite.png) -22px -71px no-repeat}div.dark_rounded .pp_bottom .pp_left{background:url(../images/dark_rounded/sprite.png) -88px -80px no-repeat}div.dark_rounded .pp_bottom .pp_right{background:url(../images/dark_rounded/sprite.png) -110px -80px no-repeat}div.dark_rounded .pp_loaderIcon{background:url(../images/dark_rounded/loader.gif) center center no-repeat}div.dark_square .pp_left,div.dark_square .pp_middle,div.dark_square .pp_right,div.dark_square .pp_content{background:#000}div.dark_square .pp_description{color:#fff;margin:0 85px 0 0}div.dark_square .pp_loaderIcon{background:url(../images/dark_square/loader.gif) center center no-repeat}div.dark_square .pp_expand{background:url(../images/dark_square/sprite.png) -31px -26px no-repeat;cursor:pointer}div.dark_square .pp_expand:hover{background:url(../images/dark_square/sprite.png) -31px -47px no-repeat;cursor:pointer}div.dark_square .pp_contract{background:url(../images/dark_square/sprite.png) 0 -26px no-repeat;cursor:pointer}div.dark_square .pp_contract:hover{background:url(../images/dark_square/sprite.png) 0 -47px no-repeat;cursor:pointer}div.dark_square .pp_close{background:url(../images/dark_square/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:75px}div.dark_square .pp_nav{clear:none}div.dark_square .pp_nav .pp_play{background:url(../images/dark_square/sprite.png) -1px -100px no-repeat;height:15px;width:14px}div.dark_square .pp_nav .pp_pause{background:url(../images/dark_square/sprite.png) -24px -100px no-repeat;height:15px;width:14px}div.dark_square .pp_arrow_previous{background:url(../images/dark_square/sprite.png) 0 -71px no-repeat}div.dark_square .pp_arrow_next{background:url(../images/dark_square/sprite.png) -22px -71px no-repeat}div.dark_square .pp_next:hover{background:url(../images/dark_square/btnNext.png) center right no-repeat;cursor:pointer}div.dark_square .pp_previous:hover{background:url(../images/dark_square/btnPrevious.png) center left no-repeat;cursor:pointer}div.light_square .pp_expand{background:url(../images/light_square/sprite.png) -31px -26px no-repeat;cursor:pointer}div.light_square .pp_expand:hover{background:url(../images/light_square/sprite.png) -31px -47px no-repeat;cursor:pointer}div.light_square .pp_contract{background:url(../images/light_square/sprite.png) 0 -26px no-repeat;cursor:pointer}div.light_square .pp_contract:hover{background:url(../images/light_square/sprite.png) 0 -47px no-repeat;cursor:pointer}div.light_square .pp_close{background:url(../images/light_square/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:75px}div.light_square .pp_nav .pp_play{background:url(../images/light_square/sprite.png) -1px -100px no-repeat;height:15px;width:14px}div.light_square .pp_nav .pp_pause{background:url(../images/light_square/sprite.png) -24px -100px no-repeat;height:15px;width:14px}div.light_square .pp_arrow_previous{background:url(../images/light_square/sprite.png) 0 -71px no-repeat}div.light_square .pp_arrow_next{background:url(../images/light_square/sprite.png) -22px -71px no-repeat}div.light_square .pp_next:hover{background:url(../images/light_square/btnNext.png) center right no-repeat;cursor:pointer}div.light_square .pp_previous:hover{background:url(../images/light_square/btnPrevious.png) center left no-repeat;cursor:pointer}div.facebook .pp_top .pp_left{background:url(../images/facebook/sprite.png) -88px -53px no-repeat}div.facebook .pp_top .pp_middle{background:url(../images/facebook/contentPatternTop.png) top left repeat-x}div.facebook .pp_top .pp_right{background:url(../images/facebook/sprite.png) -110px -53px no-repeat}div.facebook .pp_content_container .pp_left{background:url(../images/facebook/contentPatternLeft.png) top left repeat-y}div.facebook .pp_content_container .pp_right{background:url(../images/facebook/contentPatternRight.png) top right repeat-y}div.facebook .pp_expand{background:url(../images/facebook/sprite.png) -31px -26px no-repeat;cursor:pointer}div.facebook .pp_expand:hover{background:url(../images/facebook/sprite.png) -31px -47px no-repeat;cursor:pointer}div.facebook .pp_contract{background:url(../images/facebook/sprite.png) 0 -26px no-repeat;cursor:pointer}div.facebook .pp_contract:hover{background:url(../images/facebook/sprite.png) 0 -47px no-repeat;cursor:pointer}div.facebook .pp_close{background:url(../images/facebook/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:22px}div.facebook .pp_description{margin:0 37px 0 0}div.facebook .pp_loaderIcon{background:url(../images/facebook/loader.gif) center center no-repeat}div.facebook .pp_arrow_previous{background:url(../images/facebook/sprite.png) 0 -71px no-repeat;height:22px;margin-top:0;width:22px}div.facebook .pp_arrow_previous.disabled{background-position:0 -96px;cursor:default}div.facebook .pp_arrow_next{background:url(../images/facebook/sprite.png) -32px -71px no-repeat;height:22px;margin-top:0;width:22px}div.facebook .pp_arrow_next.disabled{background-position:-32px -96px;cursor:default}div.facebook .pp_nav{margin-top:0}div.facebook .pp_nav p{font-size:15px;padding:0 3px 0 4px}div.facebook .pp_nav .pp_play{background:url(../images/facebook/sprite.png) -1px -123px no-repeat;height:22px;width:22px}div.facebook .pp_nav .pp_pause{background:url(../images/facebook/sprite.png) -32px -123px no-repeat;height:22px;width:22px}div.facebook .pp_next:hover{background:url(../images/facebook/btnNext.png) center right no-repeat;cursor:pointer}div.facebook .pp_previous:hover{background:url(../images/facebook/btnPrevious.png) center left no-repeat;cursor:pointer}div.facebook .pp_bottom .pp_left{background:url(../images/facebook/sprite.png) -88px -80px no-repeat}div.facebook .pp_bottom .pp_middle{background:url(../images/facebook/contentPatternBottom.png) top left repeat-x}div.facebook .pp_bottom .pp_right{background:url(../images/facebook/sprite.png) -110px -80px no-repeat}div.pp_pic_holder a:focus{outline:0}div.pp_overlay{background:#000;display:none;left:0;position:absolute;top:0;width:100%;z-index:9500}div.pp_pic_holder{display:none;position:absolute;width:100px;z-index:10000}.pp_content{height:40px;min-width:40px}* html .pp_content{width:40px}.pp_content_container{position:relative;text-align:left;width:100%}.pp_content_container .pp_left{padding-left:20px}.pp_content_container .pp_right{padding-right:20px}.pp_content_container .pp_details{float:left;margin:10px 0 2px}.pp_description{display:none;margin:0}.pp_social{float:left;margin:0}.pp_social .facebook{float:left;margin-left:5px;overflow:hidden;width:55px}.pp_social .twitter{float:left}.pp_nav{clear:right;float:left;margin:3px 10px 0 0}.pp_nav p{float:left;margin:2px 4px;white-space:nowrap}.pp_nav .pp_play,.pp_nav .pp_pause{float:left;margin-right:4px;text-indent:-10000px}a.pp_arrow_previous,a.pp_arrow_next{display:block;float:left;height:15px;margin-top:3px;overflow:hidden;text-indent:-10000px;width:14px}.pp_hoverContainer{position:absolute;top:0;width:100%;z-index:2000}.pp_gallery{display:none;left:50%;margin-top:-50px;position:absolute;z-index:10000}.pp_gallery div{float:left;overflow:hidden;position:relative}.pp_gallery ul{float:left;height:35px;margin:0 0 0 5px;padding:0;position:relative;white-space:nowrap}.pp_gallery ul a{border:1px rgba(0,0,0,.5) solid;display:block;float:left;height:33px;overflow:hidden}.pp_gallery ul a img{border:0}.pp_gallery li{display:block;float:left;margin:0 5px 0 0;padding:0}.pp_gallery li.default a{background:url(../images/facebook/default_thumbnail.gif) 0 0 no-repeat;display:block;height:33px;width:50px}.pp_gallery .pp_arrow_previous,.pp_gallery .pp_arrow_next{margin-top:7px!important}a.pp_next{background:url(../images/light_rounded/btnNext.png) 10000px 10000px no-repeat;display:block;float:right;height:100%;text-indent:-10000px;width:49%}a.pp_previous{background:url(../images/light_rounded/btnNext.png) 10000px 10000px no-repeat;display:block;float:left;height:100%;text-indent:-10000px;width:49%}a.pp_expand,a.pp_contract{cursor:pointer;display:none;height:20px;position:absolute;right:30px;text-indent:-10000px;top:10px;width:20px;z-index:20000}a.pp_close{display:block;line-height:22px;position:absolute;right:0;text-indent:-10000px;top:0}.pp_loaderIcon{display:block;height:24px;left:50%;margin:-12px 0 0 -12px;position:absolute;top:50%;width:24px}#pp_full_res{line-height:1!important}#pp_full_res .pp_inline{text-align:left}#pp_full_res .pp_inline p{margin:0 0 15px}div.ppt{color:#fff;display:none;font-size:17px;margin:0 0 5px 15px;z-index:9999}div.pp_default .pp_content,div.light_rounded .pp_content{background-color:#fff}div.pp_default #pp_full_res .pp_inline,div.light_rounded .pp_content .ppt,div.light_rounded #pp_full_res .pp_inline,div.light_square .pp_content .ppt,div.light_square #pp_full_res .pp_inline,div.facebook .pp_content .ppt,div.facebook #pp_full_res .pp_inline{color:#000}div.pp_default .pp_gallery ul li a:hover,div.pp_default .pp_gallery ul li.selected a,.pp_gallery ul a:hover,.pp_gallery li.selected a{border-color:#fff}div.pp_default .pp_details,div.light_rounded .pp_details,div.dark_rounded .pp_details,div.dark_square .pp_details,div.light_square .pp_details,div.facebook .pp_details{position:relative}div.light_rounded .pp_top .pp_middle,div.light_rounded .pp_content_container .pp_left,div.light_rounded .pp_content_container .pp_right,div.light_rounded .pp_bottom .pp_middle,div.light_square .pp_left,div.light_square .pp_middle,div.light_square .pp_right,div.light_square .pp_content,div.facebook .pp_content{background:#fff}div.light_rounded .pp_description,div.light_square .pp_description{margin-right:85px}div.light_rounded .pp_gallery a.pp_arrow_previous,div.light_rounded .pp_gallery a.pp_arrow_next,div.dark_rounded .pp_gallery a.pp_arrow_previous,div.dark_rounded .pp_gallery a.pp_arrow_next,div.dark_square .pp_gallery a.pp_arrow_previous,div.dark_square .pp_gallery a.pp_arrow_next,div.light_square .pp_gallery a.pp_arrow_previous,div.light_square .pp_gallery a.pp_arrow_next{margin-top:12px!important}div.light_rounded .pp_arrow_previous.disabled,div.dark_rounded .pp_arrow_previous.disabled,div.dark_square .pp_arrow_previous.disabled,div.light_square .pp_arrow_previous.disabled{background-position:0 -87px;cursor:default}div.light_rounded .pp_arrow_next.disabled,div.dark_rounded .pp_arrow_next.disabled,div.dark_square .pp_arrow_next.disabled,div.light_square .pp_arrow_next.disabled{background-position:-22px -87px;cursor:default}div.light_rounded .pp_loaderIcon,div.light_square .pp_loaderIcon{background:url(../images/light_rounded/loader.gif) center center no-repeat}div.dark_rounded .pp_top .pp_middle,div.dark_rounded .pp_content,div.dark_rounded .pp_bottom .pp_middle{background:url(../images/dark_rounded/contentPattern.png) top left repeat}div.dark_rounded .currentTextHolder,div.dark_square .currentTextHolder{color:#c4c4c4}div.dark_rounded #pp_full_res .pp_inline,div.dark_square #pp_full_res .pp_inline{color:#fff}.pp_top,.pp_bottom{height:20px;position:relative}* html .pp_top,* html .pp_bottom{padding:0 20px}.pp_top .pp_left,.pp_bottom .pp_left{height:20px;left:0;position:absolute;width:20px}.pp_top .pp_middle,.pp_bottom .pp_middle{height:20px;left:20px;position:absolute;right:20px}* html .pp_top .pp_middle,* html .pp_bottom .pp_middle{left:0;position:static}.pp_top .pp_right,.pp_bottom .pp_right{height:20px;left:auto;position:absolute;right:0;top:0;width:20px}.pp_fade,.pp_gallery li.default a img{display:none}<br />
<br />
</style><br />
<script type="text/javascript"><br />
<br />
/*<br />
* Superfish v1.4.8 - jQuery menu widget<br />
* Copyright (c) 2008 Joel Birch<br />
*<br />
* Dual licensed under the MIT and GPL licenses:<br />
* http://www.opensource.org/licenses/mit-license.php<br />
* http://www.gnu.org/licenses/gpl.html<br />
*<br />
* CHANGELOG: http://users.tpg.com.au/j_birch/plugins/superfish/changelog.txt<br />
*/<br />
<br />
;(function($){<br />
$.fn.superfish = function(op){<br />
<br />
var sf = $.fn.superfish,<br />
c = sf.c,<br />
$arrow = $(['<span class="',c.arrowClass,'"> &#187;</span>'].join('')),<br />
over = function(){<br />
var $$ = $(this), menu = getMenu($$);<br />
clearTimeout(menu.sfTimer);<br />
$$.showSuperfishUl().siblings().hideSuperfishUl();<br />
},<br />
out = function(){<br />
var $$ = $(this), menu = getMenu($$), o = sf.op;<br />
clearTimeout(menu.sfTimer);<br />
menu.sfTimer=setTimeout(function(){<br />
o.retainPath=($.inArray($$[0],o.$path)>-1);<br />
$$.hideSuperfishUl();<br />
//if (o.$path.length &amp;&amp; $$.parents(['li.',o.hoverClass].join('')).length<1){over.call(o.$path);}<br />
if (o.$path.length) {<br />
if ($$.parents(['li.',o.hoverClass].join('')).length<1){over.call(o.$path);}<br />
}<br />
},o.delay); <br />
},<br />
getMenu = function($menu){<br />
var menu = $menu.parents(['ul.',c.menuClass,':first'].join(''))[0];<br />
sf.op = sf.o[menu.serial];<br />
return menu;<br />
},<br />
addArrow = function($a){ $a.addClass(c.anchorClass).append($arrow.clone()); };<br />
<br />
return this.each(function() {<br />
var s = this.serial = sf.o.length;<br />
var o = $.extend({},sf.defaults,op);<br />
o.$path = $('li.'+o.pathClass,this).slice(0,o.pathLevels).each(function(){<br />
$(this).addClass([o.hoverClass,c.bcClass].join(' '))<br />
.filter('li:has(ul)').removeClass(o.pathClass);<br />
});<br />
sf.o[s] = sf.op = o;<br />
var bcheck = false;<br />
if ($.fn.hoverIntent) {<br />
if (!o.disableHI) bcheck = true;<br />
}<br />
<br />
$('li:has(ul)',this)[(bcheck) ? 'hoverIntent' : 'hover'](over,out).each(function() {<br />
if (o.autoArrows) addArrow( $('>a:first-child',this) );<br />
})<br />
.not('.'+c.bcClass)<br />
.hideSuperfishUl();<br />
<br />
var $a = $('a',this);<br />
$a.each(function(i){<br />
var $li = $a.eq(i).parents('li');<br />
$a.eq(i).focus(function(){over.call($li);}).blur(function(){out.call($li);});<br />
});<br />
o.onInit.call(this);<br />
<br />
}).each(function() {<br />
var menuClasses = [c.menuClass];<br />
//if (sf.op.dropShadows &amp;&amp; !($.browser.msie &amp;&amp; $.browser.version < 7)) menuClasses.push(c.shadowClass);<br />
if (sf.op.dropShadows) if (!$.browser.msie) if (!($.browser.version < 7)) menuClasses.push(c.shadowClass);<br />
$(this).addClass(menuClasses.join(' '));<br />
});<br />
};<br />
<br />
var sf = $.fn.superfish;<br />
sf.o = [];<br />
sf.op = {};<br />
sf.IE7fix = function(){<br />
var o = sf.op;<br />
//if ($.browser.msie &amp;&amp; $.browser.version > 6 &amp;&amp; o.dropShadows &amp;&amp; o.animation.opacity!=undefined)<br />
if ($.browser.msie) if($.browser.version > 6) if (o.dropShadows) if (o.animation.opacity!=undefined)<br />
this.toggleClass(sf.c.shadowClass+'-off');<br />
};<br />
sf.c = {<br />
bcClass : 'sf-breadcrumb',<br />
menuClass : 'sf-js-enabled',<br />
anchorClass : 'sf-with-ul',<br />
arrowClass : 'sf-sub-indicator',<br />
shadowClass : 'sf-shadow'<br />
};<br />
sf.defaults = {<br />
hoverClass : 'sfHover',<br />
pathClass : 'overideThisToUse',<br />
pathLevels : 1,<br />
delay : 800,<br />
animation : {opacity:'show'},<br />
speed : 'normal',<br />
autoArrows : true,<br />
dropShadows : true,<br />
disableHI : false, // true disables hoverIntent detection<br />
onInit : function(){}, // callback functions<br />
onBeforeShow: function(){},<br />
onShow : function(){},<br />
onHide : function(){}<br />
};<br />
$.fn.extend({<br />
hideSuperfishUl : function(){<br />
var o = sf.op,<br />
not = (o.retainPath===true) ? o.$path : '';<br />
o.retainPath = false;<br />
var $ul = $(['li.',o.hoverClass].join(''),this).add(this).not(not).removeClass(o.hoverClass)<br />
.find('>ul').hide().css('visibility','hidden');<br />
o.onHide.call($ul);<br />
return this;<br />
},<br />
showSuperfishUl : function(){<br />
var o = sf.op,<br />
sh = sf.c.shadowClass+'-off',<br />
$ul = this.addClass(o.hoverClass)<br />
.find('>ul:hidden').css('visibility','visible');<br />
sf.IE7fix.call($ul);<br />
o.onBeforeShow.call($ul);<br />
$ul.animate(o.animation,o.speed,function(){ sf.IE7fix.call($ul); o.onShow.call($ul); });<br />
return this;<br />
}<br />
});<br />
<br />
})(jQuery);<br />
<br />
/*<br />
* Supersubs v0.2b - jQuery plugin<br />
* Copyright (c) 2008 Joel Birch<br />
*<br />
* Dual licensed under the MIT and GPL licenses:<br />
* http://www.opensource.org/licenses/mit-license.php<br />
* http://www.gnu.org/licenses/gpl.html<br />
*<br />
*<br />
* This plugin automatically adjusts submenu widths of suckerfish-style menus to that of<br />
* their longest list item children. If you use this, please expect bugs and report them<br />
* to the jQuery Google Group with the word 'Superfish' in the subject line.<br />
*<br />
*/<br />
<br />
(function($){ // $ will refer to jQuery within this closure<br />
<br />
$.fn.supersubs = function(options){<br />
var opts = $.extend({}, $.fn.supersubs.defaults, options);<br />
// return original object to support chaining<br />
return this.each(function() {<br />
// cache selections<br />
var $$ = $(this);<br />
// support metadata<br />
var o = $.meta ? $.extend({}, opts, $$.data()) : opts;<br />
// get the font size of menu.<br />
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<h1>Project Overview</h1><br />
<p>We studied the efficiency of rho-independent transcription terminators which are essential parts of synthetic genetic circuits using both theoretical calculation and experimental methods.<br />
We first developed a theoretical model. This model calculates the free energy of RNA folding and predicts the secondary structure of terminators. Based on the secondary structure, we developed an algorithm that predicts the efficiency of the terminator. The software (TTEC) based on the algorithm is available at <a href="http://www.terminatorefficiency.com">www.terminatorefficiency.com </a>(Figure.1). We also collected existing data from various publications and built up a database for comprehensive study on terminator efficiency (Figure.2). </p><br />
<img src=" https://static.igem.org/mediawiki/2012/f/fb/Proover-1.png" alt="" class="img_fl img_border" width="400px" height="200px"/><br />
<p>Figure 1, TTEC website</p><br />
<img src="https://static.igem.org/mediawiki/2012/0/07/Proover-2.png" alt="" class="img_fl img_border" width="400px" height="200px"/><br />
<p>Figure 2, Terminator Efficiency database</p><br />
<p>To validate our theoretical prediction, we performed biological experiments. We constructed a vector expressing RFP and GFP and designed 100 terminators to be inserted between RFP and GFP sequences (Figure 3). We measured the fluorescence intensity of RFP and GRP, respectively, which were further used to calculate the efficiency of the inserted terminator. We found that the experimental results are in a good agreement with theoretical prediction, and our model is useful for studying transcriptional regulation. Our terminator efficiency measurement protocol has been submitted to Biobrick foundation as a technical standard(BBF RFC 90). </p><br />
<img src="https://static.igem.org/mediawiki/2012/8/88/Proover-3.jpg" alt="" class="img_fl img_border" width="400px" height="200px"/><br />
<p>Figure 3: Plasmid design</p><br />
<p>TTEC is the first published software to predict the efficiency of terminators. Our work will help to characterize the transcription regulation and predict gene expression.</p><br />
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</html></div>M.B.ZHOUhttp://2012.igem.org/Team:SUSTC-Shenzhen-B/protocol1Team:SUSTC-Shenzhen-B/protocol12012-10-26T18:12:13Z<p>M.B.ZHOU: </p>
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<br />
/* prettyPhoto.css */<br />
div.pp_default .pp_top,div.pp_default .pp_top .pp_middle,div.pp_default .pp_top .pp_left,div.pp_default .pp_top .pp_right,div.pp_default .pp_bottom,div.pp_default .pp_bottom .pp_left,div.pp_default .pp_bottom .pp_middle,div.pp_default .pp_bottom .pp_right{height:13px}div.pp_default .pp_top .pp_left{background:url(../images/default/sprite.png) -78px -93px no-repeat}div.pp_default .pp_top .pp_middle{background:url(../images/default/sprite_x.png) top left repeat-x}div.pp_default .pp_top .pp_right{background:url(../images/default/sprite.png) -112px -93px no-repeat}div.pp_default .pp_content .ppt{color:#f8f8f8}div.pp_default .pp_content_container .pp_left{background:url(../images/default/sprite_y.png) -7px 0 repeat-y;padding-left:13px}div.pp_default .pp_content_container .pp_right{background:url(../images/default/sprite_y.png) top right repeat-y;padding-right:13px}div.pp_default .pp_next:hover{background:url(../images/default/sprite_next.png) center right no-repeat;cursor:pointer}div.pp_default .pp_previous:hover{background:url(../images/default/sprite_prev.png) center left no-repeat;cursor:pointer}div.pp_default .pp_expand{background:url(../images/default/sprite.png) 0 -29px no-repeat;cursor:pointer;height:28px;width:28px}div.pp_default .pp_expand:hover{background:url(../images/default/sprite.png) 0 -56px no-repeat;cursor:pointer}div.pp_default .pp_contract{background:url(../images/default/sprite.png) 0 -84px no-repeat;cursor:pointer;height:28px;width:28px}div.pp_default .pp_contract:hover{background:url(../images/default/sprite.png) 0 -113px no-repeat;cursor:pointer}div.pp_default .pp_close{background:url(../images/default/sprite.png) 2px 1px no-repeat;cursor:pointer;height:30px;width:30px}div.pp_default .pp_gallery ul li a{background:url(../images/default/default_thumb.png) center center #f8f8f8;border:1px solid #aaa}div.pp_default .pp_social{margin-top:7px}div.pp_default .pp_gallery a.pp_arrow_previous,div.pp_default .pp_gallery a.pp_arrow_next{left:auto;position:static}div.pp_default .pp_nav .pp_play,div.pp_default .pp_nav .pp_pause{background:url(../images/default/sprite.png) -51px 1px no-repeat;height:30px;width:30px}div.pp_default .pp_nav .pp_pause{background-position:-51px -29px}div.pp_default a.pp_arrow_previous,div.pp_default a.pp_arrow_next{background:url(../images/default/sprite.png) -31px -3px no-repeat;height:20px;margin:4px 0 0;width:20px}div.pp_default a.pp_arrow_next{background-position:-82px -3px;left:52px}div.pp_default .pp_content_container .pp_details{margin-top:5px}div.pp_default .pp_nav{clear:none;height:30px;position:relative;width:110px}div.pp_default .pp_nav .currentTextHolder{color:#999;font-family:Georgia;font-size:11px;font-style:italic;left:75px;line-height:25px;margin:0;padding:0 0 0 10px;position:absolute;top:2px}div.pp_default .pp_close:hover,div.pp_default .pp_nav .pp_play:hover,div.pp_default .pp_nav .pp_pause:hover,div.pp_default .pp_arrow_next:hover,div.pp_default .pp_arrow_previous:hover{opacity:.7}div.pp_default .pp_description{font-size:11px;font-weight:700;line-height:14px;margin:5px 50px 5px 0}div.pp_default .pp_bottom .pp_left{background:url(../images/default/sprite.png) -78px -127px no-repeat}div.pp_default .pp_bottom .pp_middle{background:url(../images/default/sprite_x.png) bottom left repeat-x}div.pp_default .pp_bottom .pp_right{background:url(../images/default/sprite.png) -112px -127px no-repeat}div.pp_default .pp_loaderIcon{background:url(../images/default/loader.gif) center center no-repeat}div.light_rounded .pp_top .pp_left{background:url(../images/light_rounded/sprite.png) -88px -53px no-repeat}div.light_rounded .pp_top .pp_right{background:url(../images/light_rounded/sprite.png) -110px -53px no-repeat}div.light_rounded .pp_next:hover{background:url(../images/light_rounded/btnNext.png) center right no-repeat;cursor:pointer}div.light_rounded .pp_previous:hover{background:url(../images/light_rounded/btnPrevious.png) center left no-repeat;cursor:pointer}div.light_rounded .pp_expand{background:url(../images/light_rounded/sprite.png) -31px -26px no-repeat;cursor:pointer}div.light_rounded .pp_expand:hover{background:url(../images/light_rounded/sprite.png) -31px -47px no-repeat;cursor:pointer}div.light_rounded .pp_contract{background:url(../images/light_rounded/sprite.png) 0 -26px no-repeat;cursor:pointer}div.light_rounded .pp_contract:hover{background:url(../images/light_rounded/sprite.png) 0 -47px no-repeat;cursor:pointer}div.light_rounded .pp_close{background:url(../images/light_rounded/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:75px}div.light_rounded .pp_nav .pp_play{background:url(../images/light_rounded/sprite.png) -1px -100px no-repeat;height:15px;width:14px}div.light_rounded .pp_nav .pp_pause{background:url(../images/light_rounded/sprite.png) -24px -100px no-repeat;height:15px;width:14px}div.light_rounded .pp_arrow_previous{background:url(../images/light_rounded/sprite.png) 0 -71px no-repeat}div.light_rounded .pp_arrow_next{background:url(../images/light_rounded/sprite.png) -22px -71px no-repeat}div.light_rounded .pp_bottom .pp_left{background:url(../images/light_rounded/sprite.png) -88px -80px no-repeat}div.light_rounded .pp_bottom .pp_right{background:url(../images/light_rounded/sprite.png) -110px -80px no-repeat}div.dark_rounded .pp_top .pp_left{background:url(../images/dark_rounded/sprite.png) -88px -53px no-repeat}div.dark_rounded .pp_top .pp_right{background:url(../images/dark_rounded/sprite.png) -110px -53px no-repeat}div.dark_rounded .pp_content_container .pp_left{background:url(../images/dark_rounded/contentPattern.png) top left repeat-y}div.dark_rounded .pp_content_container .pp_right{background:url(../images/dark_rounded/contentPattern.png) top right repeat-y}div.dark_rounded .pp_next:hover{background:url(../images/dark_rounded/btnNext.png) center right no-repeat;cursor:pointer}div.dark_rounded .pp_previous:hover{background:url(../images/dark_rounded/btnPrevious.png) center left no-repeat;cursor:pointer}div.dark_rounded .pp_expand{background:url(../images/dark_rounded/sprite.png) -31px -26px no-repeat;cursor:pointer}div.dark_rounded .pp_expand:hover{background:url(../images/dark_rounded/sprite.png) -31px -47px no-repeat;cursor:pointer}div.dark_rounded .pp_contract{background:url(../images/dark_rounded/sprite.png) 0 -26px no-repeat;cursor:pointer}div.dark_rounded .pp_contract:hover{background:url(../images/dark_rounded/sprite.png) 0 -47px no-repeat;cursor:pointer}div.dark_rounded .pp_close{background:url(../images/dark_rounded/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:75px}div.dark_rounded .pp_description{color:#fff;margin-right:85px}div.dark_rounded .pp_nav .pp_play{background:url(../images/dark_rounded/sprite.png) -1px -100px no-repeat;height:15px;width:14px}div.dark_rounded .pp_nav .pp_pause{background:url(../images/dark_rounded/sprite.png) -24px -100px no-repeat;height:15px;width:14px}div.dark_rounded .pp_arrow_previous{background:url(../images/dark_rounded/sprite.png) 0 -71px no-repeat}div.dark_rounded .pp_arrow_next{background:url(../images/dark_rounded/sprite.png) -22px -71px no-repeat}div.dark_rounded .pp_bottom .pp_left{background:url(../images/dark_rounded/sprite.png) -88px -80px no-repeat}div.dark_rounded .pp_bottom .pp_right{background:url(../images/dark_rounded/sprite.png) -110px -80px no-repeat}div.dark_rounded .pp_loaderIcon{background:url(../images/dark_rounded/loader.gif) center center no-repeat}div.dark_square .pp_left,div.dark_square .pp_middle,div.dark_square .pp_right,div.dark_square .pp_content{background:#000}div.dark_square .pp_description{color:#fff;margin:0 85px 0 0}div.dark_square .pp_loaderIcon{background:url(../images/dark_square/loader.gif) center center no-repeat}div.dark_square .pp_expand{background:url(../images/dark_square/sprite.png) -31px -26px no-repeat;cursor:pointer}div.dark_square .pp_expand:hover{background:url(../images/dark_square/sprite.png) -31px -47px no-repeat;cursor:pointer}div.dark_square .pp_contract{background:url(../images/dark_square/sprite.png) 0 -26px no-repeat;cursor:pointer}div.dark_square .pp_contract:hover{background:url(../images/dark_square/sprite.png) 0 -47px no-repeat;cursor:pointer}div.dark_square .pp_close{background:url(../images/dark_square/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:75px}div.dark_square .pp_nav{clear:none}div.dark_square .pp_nav .pp_play{background:url(../images/dark_square/sprite.png) -1px -100px no-repeat;height:15px;width:14px}div.dark_square .pp_nav .pp_pause{background:url(../images/dark_square/sprite.png) -24px -100px no-repeat;height:15px;width:14px}div.dark_square .pp_arrow_previous{background:url(../images/dark_square/sprite.png) 0 -71px no-repeat}div.dark_square .pp_arrow_next{background:url(../images/dark_square/sprite.png) -22px -71px no-repeat}div.dark_square .pp_next:hover{background:url(../images/dark_square/btnNext.png) center right no-repeat;cursor:pointer}div.dark_square .pp_previous:hover{background:url(../images/dark_square/btnPrevious.png) center left no-repeat;cursor:pointer}div.light_square .pp_expand{background:url(../images/light_square/sprite.png) -31px -26px no-repeat;cursor:pointer}div.light_square .pp_expand:hover{background:url(../images/light_square/sprite.png) -31px -47px no-repeat;cursor:pointer}div.light_square .pp_contract{background:url(../images/light_square/sprite.png) 0 -26px no-repeat;cursor:pointer}div.light_square .pp_contract:hover{background:url(../images/light_square/sprite.png) 0 -47px no-repeat;cursor:pointer}div.light_square .pp_close{background:url(../images/light_square/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:75px}div.light_square .pp_nav .pp_play{background:url(../images/light_square/sprite.png) -1px -100px no-repeat;height:15px;width:14px}div.light_square .pp_nav .pp_pause{background:url(../images/light_square/sprite.png) -24px -100px no-repeat;height:15px;width:14px}div.light_square .pp_arrow_previous{background:url(../images/light_square/sprite.png) 0 -71px no-repeat}div.light_square .pp_arrow_next{background:url(../images/light_square/sprite.png) -22px -71px no-repeat}div.light_square .pp_next:hover{background:url(../images/light_square/btnNext.png) center right no-repeat;cursor:pointer}div.light_square .pp_previous:hover{background:url(../images/light_square/btnPrevious.png) center left no-repeat;cursor:pointer}div.facebook .pp_top .pp_left{background:url(../images/facebook/sprite.png) -88px -53px no-repeat}div.facebook .pp_top .pp_middle{background:url(../images/facebook/contentPatternTop.png) top left repeat-x}div.facebook .pp_top .pp_right{background:url(../images/facebook/sprite.png) -110px -53px no-repeat}div.facebook .pp_content_container .pp_left{background:url(../images/facebook/contentPatternLeft.png) top left repeat-y}div.facebook .pp_content_container .pp_right{background:url(../images/facebook/contentPatternRight.png) top right repeat-y}div.facebook .pp_expand{background:url(../images/facebook/sprite.png) -31px -26px no-repeat;cursor:pointer}div.facebook .pp_expand:hover{background:url(../images/facebook/sprite.png) -31px -47px no-repeat;cursor:pointer}div.facebook .pp_contract{background:url(../images/facebook/sprite.png) 0 -26px no-repeat;cursor:pointer}div.facebook .pp_contract:hover{background:url(../images/facebook/sprite.png) 0 -47px no-repeat;cursor:pointer}div.facebook .pp_close{background:url(../images/facebook/sprite.png) -1px -1px no-repeat;cursor:pointer;height:22px;width:22px}div.facebook .pp_description{margin:0 37px 0 0}div.facebook .pp_loaderIcon{background:url(../images/facebook/loader.gif) center center no-repeat}div.facebook .pp_arrow_previous{background:url(../images/facebook/sprite.png) 0 -71px no-repeat;height:22px;margin-top:0;width:22px}div.facebook .pp_arrow_previous.disabled{background-position:0 -96px;cursor:default}div.facebook .pp_arrow_next{background:url(../images/facebook/sprite.png) -32px -71px no-repeat;height:22px;margin-top:0;width:22px}div.facebook .pp_arrow_next.disabled{background-position:-32px -96px;cursor:default}div.facebook .pp_nav{margin-top:0}div.facebook .pp_nav p{font-size:15px;padding:0 3px 0 4px}div.facebook .pp_nav .pp_play{background:url(../images/facebook/sprite.png) -1px -123px no-repeat;height:22px;width:22px}div.facebook .pp_nav .pp_pause{background:url(../images/facebook/sprite.png) -32px -123px no-repeat;height:22px;width:22px}div.facebook .pp_next:hover{background:url(../images/facebook/btnNext.png) center right no-repeat;cursor:pointer}div.facebook .pp_previous:hover{background:url(../images/facebook/btnPrevious.png) center left no-repeat;cursor:pointer}div.facebook .pp_bottom .pp_left{background:url(../images/facebook/sprite.png) -88px -80px no-repeat}div.facebook .pp_bottom .pp_middle{background:url(../images/facebook/contentPatternBottom.png) top left repeat-x}div.facebook .pp_bottom .pp_right{background:url(../images/facebook/sprite.png) -110px -80px no-repeat}div.pp_pic_holder a:focus{outline:0}div.pp_overlay{background:#000;display:none;left:0;position:absolute;top:0;width:100%;z-index:9500}div.pp_pic_holder{display:none;position:absolute;width:100px;z-index:10000}.pp_content{height:40px;min-width:40px}* html .pp_content{width:40px}.pp_content_container{position:relative;text-align:left;width:100%}.pp_content_container .pp_left{padding-left:20px}.pp_content_container .pp_right{padding-right:20px}.pp_content_container .pp_details{float:left;margin:10px 0 2px}.pp_description{display:none;margin:0}.pp_social{float:left;margin:0}.pp_social .facebook{float:left;margin-left:5px;overflow:hidden;width:55px}.pp_social .twitter{float:left}.pp_nav{clear:right;float:left;margin:3px 10px 0 0}.pp_nav p{float:left;margin:2px 4px;white-space:nowrap}.pp_nav .pp_play,.pp_nav .pp_pause{float:left;margin-right:4px;text-indent:-10000px}a.pp_arrow_previous,a.pp_arrow_next{display:block;float:left;height:15px;margin-top:3px;overflow:hidden;text-indent:-10000px;width:14px}.pp_hoverContainer{position:absolute;top:0;width:100%;z-index:2000}.pp_gallery{display:none;left:50%;margin-top:-50px;position:absolute;z-index:10000}.pp_gallery div{float:left;overflow:hidden;position:relative}.pp_gallery ul{float:left;height:35px;margin:0 0 0 5px;padding:0;position:relative;white-space:nowrap}.pp_gallery ul a{border:1px rgba(0,0,0,.5) solid;display:block;float:left;height:33px;overflow:hidden}.pp_gallery ul a img{border:0}.pp_gallery li{display:block;float:left;margin:0 5px 0 0;padding:0}.pp_gallery li.default a{background:url(../images/facebook/default_thumbnail.gif) 0 0 no-repeat;display:block;height:33px;width:50px}.pp_gallery .pp_arrow_previous,.pp_gallery .pp_arrow_next{margin-top:7px!important}a.pp_next{background:url(../images/light_rounded/btnNext.png) 10000px 10000px no-repeat;display:block;float:right;height:100%;text-indent:-10000px;width:49%}a.pp_previous{background:url(../images/light_rounded/btnNext.png) 10000px 10000px no-repeat;display:block;float:left;height:100%;text-indent:-10000px;width:49%}a.pp_expand,a.pp_contract{cursor:pointer;display:none;height:20px;position:absolute;right:30px;text-indent:-10000px;top:10px;width:20px;z-index:20000}a.pp_close{display:block;line-height:22px;position:absolute;right:0;text-indent:-10000px;top:0}.pp_loaderIcon{display:block;height:24px;left:50%;margin:-12px 0 0 -12px;position:absolute;top:50%;width:24px}#pp_full_res{line-height:1!important}#pp_full_res .pp_inline{text-align:left}#pp_full_res .pp_inline p{margin:0 0 15px}div.ppt{color:#fff;display:none;font-size:17px;margin:0 0 5px 15px;z-index:9999}div.pp_default .pp_content,div.light_rounded .pp_content{background-color:#fff}div.pp_default #pp_full_res .pp_inline,div.light_rounded .pp_content .ppt,div.light_rounded #pp_full_res .pp_inline,div.light_square .pp_content .ppt,div.light_square #pp_full_res .pp_inline,div.facebook .pp_content .ppt,div.facebook #pp_full_res .pp_inline{color:#000}div.pp_default .pp_gallery ul li a:hover,div.pp_default .pp_gallery ul li.selected a,.pp_gallery ul a:hover,.pp_gallery li.selected a{border-color:#fff}div.pp_default .pp_details,div.light_rounded .pp_details,div.dark_rounded .pp_details,div.dark_square .pp_details,div.light_square .pp_details,div.facebook .pp_details{position:relative}div.light_rounded .pp_top .pp_middle,div.light_rounded .pp_content_container .pp_left,div.light_rounded .pp_content_container .pp_right,div.light_rounded .pp_bottom .pp_middle,div.light_square .pp_left,div.light_square .pp_middle,div.light_square .pp_right,div.light_square .pp_content,div.facebook .pp_content{background:#fff}div.light_rounded .pp_description,div.light_square .pp_description{margin-right:85px}div.light_rounded .pp_gallery a.pp_arrow_previous,div.light_rounded .pp_gallery a.pp_arrow_next,div.dark_rounded .pp_gallery a.pp_arrow_previous,div.dark_rounded .pp_gallery a.pp_arrow_next,div.dark_square .pp_gallery a.pp_arrow_previous,div.dark_square .pp_gallery a.pp_arrow_next,div.light_square .pp_gallery a.pp_arrow_previous,div.light_square .pp_gallery a.pp_arrow_next{margin-top:12px!important}div.light_rounded .pp_arrow_previous.disabled,div.dark_rounded .pp_arrow_previous.disabled,div.dark_square .pp_arrow_previous.disabled,div.light_square .pp_arrow_previous.disabled{background-position:0 -87px;cursor:default}div.light_rounded .pp_arrow_next.disabled,div.dark_rounded .pp_arrow_next.disabled,div.dark_square .pp_arrow_next.disabled,div.light_square .pp_arrow_next.disabled{background-position:-22px -87px;cursor:default}div.light_rounded .pp_loaderIcon,div.light_square .pp_loaderIcon{background:url(../images/light_rounded/loader.gif) center center no-repeat}div.dark_rounded .pp_top .pp_middle,div.dark_rounded .pp_content,div.dark_rounded .pp_bottom .pp_middle{background:url(../images/dark_rounded/contentPattern.png) top left repeat}div.dark_rounded .currentTextHolder,div.dark_square .currentTextHolder{color:#c4c4c4}div.dark_rounded #pp_full_res .pp_inline,div.dark_square #pp_full_res .pp_inline{color:#fff}.pp_top,.pp_bottom{height:20px;position:relative}* html .pp_top,* html .pp_bottom{padding:0 20px}.pp_top .pp_left,.pp_bottom .pp_left{height:20px;left:0;position:absolute;width:20px}.pp_top .pp_middle,.pp_bottom .pp_middle{height:20px;left:20px;position:absolute;right:20px}* html .pp_top .pp_middle,* html .pp_bottom .pp_middle{left:0;position:static}.pp_top .pp_right,.pp_bottom .pp_right{height:20px;left:auto;position:absolute;right:0;top:0;width:20px}.pp_fade,.pp_gallery li.default a img{display:none}<br />
<br />
</style><br />
<script type="text/javascript"><br />
<br />
/*<br />
* Superfish v1.4.8 - jQuery menu widget<br />
* Copyright (c) 2008 Joel Birch<br />
*<br />
* Dual licensed under the MIT and GPL licenses:<br />
* http://www.opensource.org/licenses/mit-license.php<br />
* http://www.gnu.org/licenses/gpl.html<br />
*<br />
* CHANGELOG: http://users.tpg.com.au/j_birch/plugins/superfish/changelog.txt<br />
*/<br />
<br />
;(function($){<br />
$.fn.superfish = function(op){<br />
<br />
var sf = $.fn.superfish,<br />
c = sf.c,<br />
$arrow = $(['<span class="',c.arrowClass,'"> &#187;</span>'].join('')),<br />
over = function(){<br />
var $$ = $(this), menu = getMenu($$);<br />
clearTimeout(menu.sfTimer);<br />
$$.showSuperfishUl().siblings().hideSuperfishUl();<br />
},<br />
out = function(){<br />
var $$ = $(this), menu = getMenu($$), o = sf.op;<br />
clearTimeout(menu.sfTimer);<br />
menu.sfTimer=setTimeout(function(){<br />
o.retainPath=($.inArray($$[0],o.$path)>-1);<br />
$$.hideSuperfishUl();<br />
//if (o.$path.length &amp;&amp; $$.parents(['li.',o.hoverClass].join('')).length<1){over.call(o.$path);}<br />
if (o.$path.length) {<br />
if ($$.parents(['li.',o.hoverClass].join('')).length<1){over.call(o.$path);}<br />
}<br />
},o.delay); <br />
},<br />
getMenu = function($menu){<br />
var menu = $menu.parents(['ul.',c.menuClass,':first'].join(''))[0];<br />
sf.op = sf.o[menu.serial];<br />
return menu;<br />
},<br />
addArrow = function($a){ $a.addClass(c.anchorClass).append($arrow.clone()); };<br />
<br />
return this.each(function() {<br />
var s = this.serial = sf.o.length;<br />
var o = $.extend({},sf.defaults,op);<br />
o.$path = $('li.'+o.pathClass,this).slice(0,o.pathLevels).each(function(){<br />
$(this).addClass([o.hoverClass,c.bcClass].join(' '))<br />
.filter('li:has(ul)').removeClass(o.pathClass);<br />
});<br />
sf.o[s] = sf.op = o;<br />
var bcheck = false;<br />
if ($.fn.hoverIntent) {<br />
if (!o.disableHI) bcheck = true;<br />
}<br />
<br />
$('li:has(ul)',this)[(bcheck) ? 'hoverIntent' : 'hover'](over,out).each(function() {<br />
if (o.autoArrows) addArrow( $('>a:first-child',this) );<br />
})<br />
.not('.'+c.bcClass)<br />
.hideSuperfishUl();<br />
<br />
var $a = $('a',this);<br />
$a.each(function(i){<br />
var $li = $a.eq(i).parents('li');<br />
$a.eq(i).focus(function(){over.call($li);}).blur(function(){out.call($li);});<br />
});<br />
o.onInit.call(this);<br />
<br />
}).each(function() {<br />
var menuClasses = [c.menuClass];<br />
//if (sf.op.dropShadows &amp;&amp; !($.browser.msie &amp;&amp; $.browser.version < 7)) menuClasses.push(c.shadowClass);<br />
if (sf.op.dropShadows) if (!$.browser.msie) if (!($.browser.version < 7)) menuClasses.push(c.shadowClass);<br />
$(this).addClass(menuClasses.join(' '));<br />
});<br />
};<br />
<br />
var sf = $.fn.superfish;<br />
sf.o = [];<br />
sf.op = {};<br />
sf.IE7fix = function(){<br />
var o = sf.op;<br />
//if ($.browser.msie &amp;&amp; $.browser.version > 6 &amp;&amp; o.dropShadows &amp;&amp; o.animation.opacity!=undefined)<br />
if ($.browser.msie) if($.browser.version > 6) if (o.dropShadows) if (o.animation.opacity!=undefined)<br />
this.toggleClass(sf.c.shadowClass+'-off');<br />
};<br />
sf.c = {<br />
bcClass : 'sf-breadcrumb',<br />
menuClass : 'sf-js-enabled',<br />
anchorClass : 'sf-with-ul',<br />
arrowClass : 'sf-sub-indicator',<br />
shadowClass : 'sf-shadow'<br />
};<br />
sf.defaults = {<br />
hoverClass : 'sfHover',<br />
pathClass : 'overideThisToUse',<br />
pathLevels : 1,<br />
delay : 800,<br />
animation : {opacity:'show'},<br />
speed : 'normal',<br />
autoArrows : true,<br />
dropShadows : true,<br />
disableHI : false, // true disables hoverIntent detection<br />
onInit : function(){}, // callback functions<br />
onBeforeShow: function(){},<br />
onShow : function(){},<br />
onHide : function(){}<br />
};<br />
$.fn.extend({<br />
hideSuperfishUl : function(){<br />
var o = sf.op,<br />
not = (o.retainPath===true) ? o.$path : '';<br />
o.retainPath = false;<br />
var $ul = $(['li.',o.hoverClass].join(''),this).add(this).not(not).removeClass(o.hoverClass)<br />
.find('>ul').hide().css('visibility','hidden');<br />
o.onHide.call($ul);<br />
return this;<br />
},<br />
showSuperfishUl : function(){<br />
var o = sf.op,<br />
sh = sf.c.shadowClass+'-off',<br />
$ul = this.addClass(o.hoverClass)<br />
.find('>ul:hidden').css('visibility','visible');<br />
sf.IE7fix.call($ul);<br />
o.onBeforeShow.call($ul);<br />
$ul.animate(o.animation,o.speed,function(){ sf.IE7fix.call($ul); o.onShow.call($ul); });<br />
return this;<br />
}<br />
});<br />
<br />
})(jQuery);<br />
<br />
/*<br />
* Supersubs v0.2b - jQuery plugin<br />
* Copyright (c) 2008 Joel Birch<br />
*<br />
* Dual licensed under the MIT and GPL licenses:<br />
* http://www.opensource.org/licenses/mit-license.php<br />
* http://www.gnu.org/licenses/gpl.html<br />
*<br />
*<br />
* This plugin automatically adjusts submenu widths of suckerfish-style menus to that of<br />
* their longest list item children. If you use this, please expect bugs and report them<br />
* to the jQuery Google Group with the word 'Superfish' in the subject line.<br />
*<br />
*/<br />
<br />
(function($){ // $ will refer to jQuery within this closure<br />
<br />
$.fn.supersubs = function(options){<br />
var opts = $.extend({}, $.fn.supersubs.defaults, options);<br />
// return original object to support chaining<br />
return this.each(function() {<br />
// cache selections<br />
var $$ = $(this);<br />
// support metadata<br />
var o = $.meta ? $.extend({}, opts, $$.data()) : opts;<br />
// get the font size of menu.<br />
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<h1><p>Lab Protocol</p></h1><br />
</font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Brief Process</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p><a href="#Site-Directed_Mutagenesis">1. Site-Directed Mutagenesis</a></p><br />
<p><a href="#Restriction">2. Restriction Enzyme Digestion and Electrophoresis</a></p><br />
<p><a href="#Media">3. Media Preparation</a></p><br />
<p><a href="#Bacterial">4. Bacterial Transformation</a></p><br />
<p><a href="#Colony">5. Colony PCR for Verification</a></p><br />
<p><a href="#Culture">6. Cultivate the Bacteria</a></p><br />
<p><a href="#Plasmid">7. Plasmid DNA Isolation</a></p><br />
<p><a href="#Restriction_2">8. Restriction Enzyme Digestion and Electrophoresis</a></p><br />
<p><a href="#Amplify">9. Polymerase Chain Reaction and Electrophoresis</a></p><br />
<p><a href="#Electrophoresis">10. Double Restriction Enzyme Digestion and Electrophoresis </a></p><br />
<p><a href="#Ligation">11. Ligation</a></p><br />
<p><a href="#Bacterial_Transformation">12. Bacterial Transformation</a></p><br />
<p><a href="#bacterial_colony">13. Bacterial Colony PCR</a></p><br />
<p><a href="#Culture_the">14. Cultivate the Bacteria</a></p><br />
<p><a href="#Plasmid_DNA">15. Plasmid DNA Isolation</a></p><br />
<p><a href="#Restriction_Enzyme">16. Restriction Enzyme Digestion and Electrophoresis</a></p><br />
<p><a href="#Ligation1">17. Ligation</a></p><br />
<p><a href="#Bacteria">18. Bacteria Transformation</a></p><br />
<p><a href="#Cultivate">19. Cultivate the Bacteria</a></p><br />
<p><a href="#Flow">20. Flow Cytometer Analysis</a></p><br />
<p><a href="#Fluorescence">21. Fluorescence Microscope </a></p><br />
<br><br />
<a name="Site-Directed_Mutagenesis" ></a></font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Site-Directed Mutagenesis</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Plasmid psb1a3 is chosen to be the vector that ligates GPF and RFP fragments.To protect the structural integrity of the constructed plasmid, a restriction enzyme cutting site named Pst I need to be mutated to Afl II. Proper primers are designed for this purpose. </p><br />
<p><strong>Method</strong></p><br />
<p>1. </p><br />
</font><br />
<ul><br />
<li>Prepare the sample reaction as indicated below:</li><br />
</ul><br />
<p>Total: 25μl<br /><br />
+ 0.25 μl of Ex Taq polymerase <br /><br />
+ 2.5 μl of 10× Taq reaction buffer<br /><br />
+ 2.0 μl of dNTP(2mM) <br /><br />
+ 1.0 μl of template (E.coli plasmid 817)<br /><br />
+ 1.0 μl of oligonucleotide primer PtoA-F<br /><br />
+ 1.0 μl of oligonucleotide primer PtoA-R <br /><br />
+ 18.25 μl of ddH2O <br /><br />
Note: Here listed the primers and their sequences.<br /><br />
PtoA-F 5'-CCACCTGACGTCTAAGAAAC-3'<br /><br />
PtoA-R 5'-ATGATCATCGCCGGCGAATTCAGGC-3'&nbsp;<br /><br />
2. Set thermocycler temperatures and the time. <br /><br />
Procedures on the thermocycler are listed below:<br /><br />
① 94˚C for 5 min<br /><br />
② 30 cycle<br /><br />
a. 94˚C for 1 min<br /><br />
b. 55˚C for 1 min<br /><br />
c. 72˚C for 1 min20sec<br /><br />
③ 4℃ for 7 hours </p><br />
<font face="Arial, Helvetica"><br />
<p><br><br />
<a name="Restriction" ></a> </p><br />
</font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Restriction Enzyme Digestion for Verification</font></b></font></h3><br />
<p align="left">In the condition of restriction enzyme cutting site is mutated correctly, a special step to proof the result is in need. A restriction enzyme digestion can be executed and result can be revealed by electrophoretogram. We used restriction enzyme Afl II digestion as a sample group and Spe I digestion as a control group.<br /><br />
<strong>Method</strong><br /><br />
1. Prepare the control reaction as indicated below:<br /><br />
Total: 10μl<br /><br />
+ 0.5μl of Pst I restriction enzyme (company :Takara)<br /><br />
+ 1μl of 10XH buffer<br /><br />
+ 1μl of plasmid DNA<br /><br />
+ 7.5μl of ddH2O <br /><br />
2. Prepare the sample reaction as indicated below:<br /><br />
Total: 10μl<br /><br />
+ 0.5μl of Afl II restriction enzyme, (company :Takara)<br /><br />
+ 1μl of 10XM buffer<br /><br />
+ 1μl of plasmid DNA<br /><br />
+ 1.0μl of 0.01% BSA<br /><br />
+ 6.5μl of ddH2O<br /><br />
3. Put the tubes in 37℃ water bath for 1-2h. <br /><br />
4. Prepare electrophoresis gel by adding 0.6g agarose to 60ml TAE (1% solution,1X,diluted from 50X TAE). Pour on conical flask and cover the Conical flask sealing surface with silver paper to avoid the loss of water vapor. Place in the microwave and microwave on middle for 1 minute at a time, pulling it out and swirling until solution is homogeneous again, then repeat(BE CAREFUL to watch the solution closely when swirling–it superheats and can boil over and cause severe burns). Continue until solution is seen clear and homogeneous with no existence of solid.Add 3 μl of Gelred ( 10000X ) . <br /><br />
5. By inserting the pipette tip below the TAE liquid and into the well, add 5μl of 1kb DNA ladder solution to first (and last if desired) well, skip one well, then begin adding the 5μl of digested DNA solutions mixed with 1μl loading buffer (6X) to the wells.<br /><br />
6. Place the cover on the electrophoresis unit, plug into the power source, and turn on voltage to 120V, set time to 30 minutes, and press the start button twice,until the bubbles are seen. DNA separation can be observed as time goes on by turning off the power supply then gently removing the basin from the electrophoresis unit (be careful not to let the gel slip out of the basin) and placing on the UV transilluminator to see DNA bands. <br /><br />
7. When the desired level of separation is obtained, the basin can be placed on the transilluminator for picture taking(Of the absence of transilluminator,we use camera to take pictures with the UV light ).<br /><br />
8. Cut the gel of specific position and collect it in tubes that have measured weight. <br /><br />
9. Use DNA Gel Extraction Ki to purify the plasmid DNA mutant-psb1a3.<br /><br />
<strong>Figure</strong></p><br />
<font face="Arial, Helvetica"><br />
<p> <img src="https://static.igem.org/mediawiki/2012/5/59/11.png" alt="" class="img_fl img_border" /> </p><br />
<p>(Figure 1 : This figure shows that the site-directed mutagenesis succeed ,we successfully change a restriction enzyme cutting site named Pst I to Afl II. Lane 1 represents the plasmid mutant-psb1a3, lane 2 shows that the mutant-psb1a3 cannot be digested by restriction enzyme Spe I ,lane 3 shows that mutant-psb1a3 can be digested by restriction enzyme Afl II.) </p><br />
<br><br />
<a name="Media"></a></font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Media Preparation</font></b></font></h3><br />
<p align="left">For all experiments involving the bacterial biomass and experimentation, proper media is chosen to grow the cells. Commonly,we use Lysogeny broth media for <em>E. coli</em>. The following is the media compositions and their quantities.<br /><br />
<strong>Lysogeny Broth (LB) liquid media (1 L)</strong><br /><br />
Measure out these following: </p><br />
<ol><br />
<li>Bacto-Tryptone - 10 g</li><br />
<li>NaCl - 10 g</li><br />
<li>Yeast Extract - 5 g</li><br />
</ol><br />
<p align="left">Add ddH2O and 5mmol/L Tris Buffer in a measuring cylinder to ensure accuracy, to make a total of 1 liter and pH is 8.0. <br /><br />
<strong>Lysogeny Broth (LB) solid media (1 L)</strong><br /><br />
Measure out these following: </p><br />
<ol><br />
<li>Bacto-Tryptone - 10 g</li><br />
<li>NaCl - 10 g</li><br />
<li>Yeast Extract - 5 g</li><br />
<li>Difco Agar - 15g</li><br />
</ol><br />
<p align="left">Add ddH2O and 5mmol/L Tris Buffer in a measuring cylinder to ensure accuracy, to make a total of 1 liter and pH is 8.0. <br /><br />
<strong>Autoclaving</strong> </p><br />
<ol><br />
<ul><br />
<li>Autoclave at 121 °C for 60 minutes. After the media cooling down enough, antibiotics Ampicillin(100mg of Ampicillin per 1ml of the media) are added. At last the media are poured 15ml on each plate and become solid.Store the plate at 4℃ refrigerator. </li><br />
</ul><br />
</ol><br />
<font face="Arial, Helvetica"><br />
<p>&nbsp;</p><br />
<br><br />
<a name="Bacterial"></a></font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Bacterial Transformation</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Introduction of exogenous DNA into cells using non-viral methods is called “Transformation”.Transformation is commonly used to introduce recombinant plasmid DNA into bacterial strains which can transform naturally or can be made competitive for transformation by artificial means.<br /><br />
Depending on the expected transformation efficiency, there are two main types of competent cells that can be used for transformation.<br /><br />
1.Chemically competent cells<br /><br />
Chemically induced competent cells are calcium chloride-treated to facilitate attachment of the plasmid DNA to the competent cell membrane. During chemical transformation, the cells are heat-shocked in a water bath; which opens the pores of the cell membrane allowing entry of plasmid DNA from the buffer. <br /><br />
2.Electrocompetent cells<br /><br />
Electrocompetent cells are prepared for transformation using electroporation, a method that uses an electrical pulse to create pores through which genetic material enters the cells. This method usually has high transformation efficiency. <br /><br />
<strong>Method</strong><br /><br />
1. Take out an appropriate number of tubes that contain competent cells(100μl ) from the freezer. Immediately place the tubes on ice, so that all but the cap is surrounded by ice. Allow the cells to thaw on ice for 2-5 min.<br /><br />
2. Visually check the cells to see whether they have thawed and gently flick the cells 1-2 times to evenly resuspend the cells.<br /><br />
3. Add 10μl PCR products(mini-prep purified) to the competent cells DH-5α. Stir gently to mix and return the tube to the ice, making sure that the tube is surrounded by ice except for the cap. Repeat for additional two times for the same samples.<br /><br />
4. Incubate the tubes on ice for 30 min.<br /><br />
5. Place the tubes in a 42°C water bath for exactly 90 sec; do not shake.<br /><br />
6. Place the tubes on ice for 2 min to cool down.<br /><br />
7. Add 800 l of room temperature LB medium to each tube.<br /><br />
8. Shake the tubes vigorously at 37<a name="OLE_LINK2" id="OLE_LINK2"></a><a name="OLE_LINK1" id="OLE_LINK1">°C</a> for 45-60 min.<br /><br />
9. Centrifuge the tubes at 3K RPM for 1 min. Discard the supernatant liquor and leave 100-200 μl of the mixtures.Mix the contents and spread the whole liquid on LB agar plates containing the appropriate antibiotic ampicillin for the plasmid.<br /><br />
10. Place the plates on the bench for several min to allow excess liquid to be absorbed, and then invert and incubate overnight at 37°C (12-16 h).</p><br />
<br><br />
<a name="Colony"></a></font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Colony PCR for Verification</font> </b></font></h3><br />
<font face="Arial, Helvetica"><br />
<P>Colony PCR is used to identify and select cell colonies that have the correct plasmid inserted. The procedure is a way to do several PCR operations on cell colonies in parallel, to evaluate the results and select the corresponding good cell colonies. After an overnight growth of E.coli, we can pick up several colonies from the plate and do a colony PCR verification. Besides, the colonies we choose and should also be stored, we can incubate these colonies in one plate after every colony has been marked.<br /><br />
<strong>Method</strong><br /><br />
1. Prepare the sample reaction as indicated below:<br /><br />
Total: 25μl <br /><br />
+ 0.25 μl of Ex Taq polymerase (company:Takara)<br /><br />
+ 2.5 μl of 10× Taq reaction buffer<br /><br />
+ 1.0 μl of R-NPS-F <br /><br />
+ 1.0 μl of G-SXA-R<br /><br />
+ 1.0 μl of plasmid mutant-psb1a3<br /><br />
+ 2.0 μl of dNTP( 25mM ) <br /><br />
+ 17.25 μl of ddH2O <br /><br />
2. Procedures on the thermocycler are listed below:<br /><br />
① 94˚C for 5 min<br /><br />
② 30 cycle<br /><br />
a. 94˚C for 1 min<br /><br />
b. 55˚C for 1 min<br /><br />
c. 72˚C for 1 min20sec<br /><br />
③ 4℃ for 7 hours <br /><br />
3. Electrophorese the total system and observe the lane separation. </P><br />
<br><br />
<a name="Culture"></a></font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Cultivate the Bacteria</b></font></h3><br />
<font face="Arial, Helvetica"><p>According to the results of the PCR detection, positive colonies are chosen and transferred them to 5ml LB liquid media ( 5μl of ampicillin added) stored in 12.5ml centrifuge tubes. Put the centrifuge tubes in 37℃ gas bath overnight. </p><br />
<br><br />
<a name="Plasmid"></a><br />
<h3><b>Plasmid DNA Isolation</b></h3><br />
<p>Use E.Z.N.A.TM Plasmid Mini I to realize plasmid DNA isolation.<br /><br />
<strong>Method</strong></p><br />
<ul><br />
<ul><br />
<li>Transfer 5 ml of overnight culture into a 1.5-ml eppendorf tube labeled with group number. </li><br />
<li>Centrifuge the sample at max. speed of desk top centrifuge and RT for 1min to pellet the cells.</li><br />
<li>Discard the supernatant. Remove as much of the supernatant as possible without disturbing the cell pellet. </li><br />
<li>Repeat step 1 and 2 twice. </li><br />
<li>Resuspend the pellet completely in 250 ml of Solution I (containing RNase A) by vortexing the samples vigorously . No clumps should be visible in the tube. </li><br />
<li>Add 250 ml of Solution II and mix the sample by gently inverting the tube 4 to 6 times. Do not vortex or shake the sample vigorously. The bacterial suspension should begin to clear which have lysed the bacterial cells in this step. <strong>Warning: </strong>Do not stop here for more than five min, as the high pH hurts your DNA! </li><br />
<li>Add 350 ml of Solution III and mix by gently inverting the tube 4 to 6 times until a flocculent white precipitate forms. Do not shake vigorously, as it might break the genomic DNA. </li><br />
<li>Centrifuge at maximum speed for 10 min at room temperature to pellet the cell debris. You should see a white precipitate in the tube after the centrifugation. </li><br />
<li>While the samples are centrifuging, for each sample, label a clean HiBind Miniprep Column which is to assembled in a 2-ml collection tube</li><br />
<li>Apply the supernatants from step 8 to the columns. </li><br />
<li>Centrifuge at maximum speed for 1 min at RT. Discard the flow-through in the collection tube. </li><br />
<li>Add 500 ml of Buffer HB to wash the Hibind Miniprep Column. Centrifuge at maximum speed for 1 min at RT. Discard the flow-through in the collection tube. </li><br />
<li>Wash the column by adding 700 ml of DNA Wash Buffer diluted with absolute ethanol. Centrifuge at maximum speed for 1 min at room temperature and discard the flow-through.</li><br />
<li>Then centrifuge the tubes again for 2 min to remove all the moisture.</li><br />
<li>Place the column in a clean 1.5 ml eppendorf tube that is labeled with the plasmid name and group number. To elute the DNA, add 50 ml of Elution Buffer to the center of each column. Let the samples stand for 2 or more minutes at RT, and then centrifuge for 1 min. The sample in the centrifuge tube (bottom) is your plasmid DNA. </li><br />
<li>Discard the column and save the sample in the eppendorf tube by placing it in the freezer (-20°C). </li><br />
</ul></ul><br />
<a name="#Restriction_2" ></a> </font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Restriction Enzyme Digestion and Electrophoresis</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Because the colony PCR test is so sensitive and affect markedly by environment factors. So we do a restriction enzyme digestion to ensure that the isolated plasmid is the site-directed mutated plasmid.<br /><br />
<strong>Method</strong><br /><br />
1. Prepare the control reaction as indicated below:<br /><br />
Total: 10μl<br /><br />
+ 0.5μl of Pst I restriction enzyme(company :Takara)<br /><br />
+ 1μl of 10XH buffer<br /><br />
+ 1μl of plasmid DNA<br /><br />
+ 7.5μl of ddH2O <br /><br />
2. Prepare the sample reaction as indicated below:<br /><br />
Total: 10μl<br /><br />
+ 0.5μl of Afl II restriction enzyme, (company :Takara)<br /><br />
+ 1μl of 10XM buffer<br /><br />
+ 1μl of plasmid DNA<br /><br />
+ 1.0μl of 0.01% BSA<br /><br />
+ 6.5μl of ddH2O <br /><br />
3. Electrophorese the total system and observe the lane separation.</p><br />
<br><br />
<a name="Amplify"></a> </font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Polymerase Chain Reaction(GFP &amp; RFP) and Electrophoresis</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>GFP and RFP DNA fragments are the insert which need to be ligate to the plasmid mutant-psb1a3. Do a PCR amplification can get enough quantities for the following reactions.<br /><br />
<strong>Method </strong><br /><br />
1.Prepare the sample reaction as indicated below:<br /><br />
Total: 100μl ( PCR <a name="OLE_LINK37" id="OLE_LINK37"></a><a name="OLE_LINK36" id="OLE_LINK36">amplification</a> of GFP fragments) <br /><br />
+ 1.0μl of Taq DNA polymerase,#EP0402<br /><br />
+ 10μl of 10XTaq buffer <br /><br />
+ 10μl of MgCl2(25mM)<br /><br />
+ 10μl of dNTP(2mM)<br /><br />
+ 2μl of G-SXA-R <br /><br />
+ 2μl of G-SXA-F<br /><br />
+ 4μl of DNA template<br /><br />
+ 61μl of ddH2O <br /><br />
Total: 100μl(PCR amplification of RFP fragments) <br /><br />
+ 1.0μl of Taq DNA polymerase, EP0402<br /><br />
+ 10μl of 10XTaq buffer <br /><br />
+ 10μl of MgCl2(25mM)<br /><br />
+ 10μl of dNTP(2mM)<br /><br />
+ 2μl of R-NPS-R <br /><br />
+ 2μl of R-NPS-F<br /><br />
+ 4μl of DNA template<br /><br />
+ 61μl of ddH2O <br /><br />
Note: Here listed the sequences of primers.<br /><br />
R-NPS-F 5'-TATAGCGGCCGCCTTAAGTAAGTAAGAGTATACG-3' <br /><br />
R-NPS-R 5'-CGGAGACTAGTCTGCAGATCACATAAGTAAAGTGATAATC-3' <br /><br />
G-SXA-F 5'-CTAGACTAGTTCTAGAGGCGGACTCACTATAGA-3' <br /><br />
G-SXA-R 5'-CCGACTTAAGGGATCCTATAAACGCAG-3'<br /><br />
2.Procedures on the thermocycler are listed below:<br /><br />
① 94˚C for 5 min<br /><br />
② 30 cycle<br /><br />
a. 94˚C for 1 min<br /><br />
b. 55˚C for 1 min<br /><br />
c. 72˚C for 1 min20sec<br /><br />
③ 4℃ for 7 hours </p><br />
</font><br />
<ul><br />
<li>Use DNA Gel Extraction Kit to purify the GFP and RFP DNA fragments after the Electrophoresis.</li><br />
</ul><br />
<p align="left"><strong>Figure</strong></p><br />
<p><font face="Arial, Helvetica"><img src="https://static.igem.org/mediawiki/2012/7/72/111.png" alt="" class="img_fl img_border" /> </font></p><br />
<p>(Figure 2 : This figure shows that The PCR reaction system can amplify large quantities of GFP and RFP DNA fragments. The digestion based on the GFP and RFP DNA fragments can be done to prepare for the ligation. Lane 1 represents the template E.coli 817 can amplify the GFP and RFP DNA fragments , lane 2 represents the template E.coli 817(355.5) can also amplify the GFP and RFP DNA fragments.) <font face="Arial, Helvetica"><br><br />
<a name="Restriction_Enzyme"></a><br />
</font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Double Restriction Enzyme Digestion and Electrophoresis.</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Use specific restriction enzymes to digest plasmid mutant-psb1a3,GFP and RFP to get sticky ends and purify the DNA fragment after the Electrophoresis.<br /><br />
<strong>Method:</strong></p><br />
</font><br />
<ul><br />
<li> Digestion of plasmid mutant-psb1a3 </li><br />
</ul><br />
<p>Prepare the sample reaction as indicated below:<br /><br />
Total: 50μl <br /><br />
+ 3.0μl of Not I restriction enzyme, #ER0591<br /><br />
+ 3.0μl of Afl II restriction enzyme, #ER0831<br /><br />
+ 5.0μl of 10X buffer O <br /><br />
+ 1.0μl of mutant-psb1a3 plasmid <br /><br />
+ 37.0μl of ddH2O<br /><br />
2. Digestion of PCR product GFP<br /><br />
Prepare the sample reaction as indicated below:<br /><br />
Total: 50μl <br /><br />
+ 3.0μl of Not I restriction enzyme, #ER0591<br /><br />
+ 3.0μl of Spe I restriction enzyme, #ER1251 <br /><br />
+ 5μl of 10X buffer Tango<br /><br />
+ 10μl of PCR products GFP<br /><br />
+ 29μl of ddH2O<br /><br />
3. Digestion of PCR product RFP<br /><br />
Prepare the sample reaction as indicated below:<br /><br />
Total: 50μl <br /><br />
+ 3.0μl of Afl II restriction enzyme, #ER0831<br /><br />
+ 3.0μl of Spe I restriction enzyme, #ER1251 <br /><br />
+ 5μl of 10X buffer Tango<br /><br />
+ 10μl of PCR products RFP<br /><br />
+ 29μl of ddH2O<br /><br />
4. Put the tubes in 37℃ environment for 4-8 hours <br /><br />
5. Use DNA Gel Extraction Kit to purify the mutant-psb1a3 fragment, GFP and RFP after digestion and named them by mutant-psb1a3 (NA) ,GFP(NS) and RFP(AS) after the Electrophoresis.</p><br />
<font face="Arial, Helvetica"><br><br />
<a name="Ligayion"></a> </font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Ligation</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Ligation is the process that target DNA gene is inserted into a plasmid. Both the vector and insert are prepared to have the sticky ends. These two kinds of DNA pieces are placed in a reaction tube and the proper DNA ligase, buffer, and cofactors are added for the reaction to take place. When done properly, the ligation will result in a successful combination of the insert and plasmid into one plasmid.Based on the digestion of mutant-psb1a3,GFP and RFP DNA fragments,also ligation can be done. We ligate mutant-psb1a3 vector and sticky GFP and RFP DNA fragments to construct an new plasmid mutant-psb1a3-GR. <br /><br />
1. Prepare the control reaction as indicated below:<br /><br />
Total: 10μl <br /><br />
+ 1.0μl of plasmid mutant-psb1a3 (NA) <br /><br />
+ 3.0μl of GFP(NS) <br /><br />
+ 3.0μl of RFP(AS) <br /><br />
+ 2.0μl of T4 DNA Ligase,#EL0011 <br /><br />
+ 1.0μl of 10XT4 Ligase buffer<br /><br />
Note: GFP(NS) means the product of GFP DNA fragments digested by restriction enzyme Not I and Spe I. <br /><br />
2. Prepare the sample reaction as indicated below:<br /><br />
Total: 10μl <br /><br />
+ 1.0μl of plasmid mutant-psb1a3 (NA) <br /><br />
+ 2.0μl of T4 DNA Ligase, #EL0011 <br /><br />
+ 1.0μl of 10XT4 Ligase buffer<br /><br />
+ 6.0μl of ddH2O<br /><br />
3. Put the tubes in 22℃ water bath, react for 8-12 hours. <br><br />
<a name="Bacterial_Transformation"></a> </font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Bacterial Transformation</font></b></font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Transform the ligation products into the DH-5α competent cells, put the plate on 37℃ gas bath for 12-16 hours. </p><br />
<br><br />
<a name="Bacterial_Colony"></a> </font><br />
<h3><font face="Arial, Helvetica"><b><font color="#0000FF">Bacterial Colony PCR</font></b> </font></h3><br />
<font face="Arial, Helvetica"><br />
<p>Colony PCR is used to identify and select cell colonies that have the correct plasmid insert. This procedure is a way to do several PCR operations on cell colonies in parallel, to evaluate the results and select the corresponding good cell colonies. After an overnight growth of E.coli, we can pick up some colonies from the plate and do a colony PCR verification. Besides, the colonies we choose and should also be stored, we can incubate these colonies in one plate after every colony has been marked.<br /><br />
<strong>Method</strong><br /><br />
1. Prepare the sample reaction as indicated below:<br /><br />
Total: 20μl <br /><br />
+ 0.25 μl of Ex Taq polymerase,#EP0402 <br /><br />
+ 2.0 μl of 10× Taq reaction buffer<br /><br />
+ 1.0 μl of R-NPS-F <br /><br />
+ 1.0 μl of G-SXA-R<br /><br />
+ 5.0 μl of bacterial colony<br /><br />
+ 2.0 μl of dNTP( 25mM ) <br /><br />
+ 8.75 μl of ddH2O <br /><br />
Note: Here listed the sequence of primers.<br /><br />
R-NPS-F 5'-TATAGCGGCCGCCTTAAGTAAGTAAGAGTATACG-3' <br /><br />
R-NPS-R 5'-CGGAGACTAGTCTGCAGATCACATAAGTAAAGTGATAATC-3' <br /><br />
G-SXA-F 5'-CTAGACTAGTTCTAGAGGCGGACTCACTATAGA-3' <br /><br />
G-SXA-R 5'-CCGACTTAAGGGATCCTATAAACGCAG-3'<br /><br />
2. Procedures on the thermocycler are listed below:<br /><br />
① 94˚C for 5 min<br /><br />
② 30 cycle<br /><br />
a. 94˚C for 1 min<br /><br />
b. 55˚C for 1 min<br /><br />
c. 72˚C for 1 min20sec<br /><br />
③ 4℃ for 7 hours </p><br />
</font><br />
<ul><br />
<li> Electrophorese the total system and observe the lane separation. </li><br />
</ul><br />
<p><strong>Figure</strong></p><br />
<p> <img src="https://static.igem.org/mediawiki/2012/0/07/1111.png" alt="" class="img_fl img_border" /> </p><br />
<p>(Figure 3: The lane on the figure are 2k fragments, it shows the GFP and RFP are ligated to the vectors which was isolated from the bacterial colonies.) </p><br />
<p><font face="Arial, Helvetica"><br><br />
<a name="Culture_the"></a><br />
</font></p><br />
<font face="Arial, Helvetica"><br />
</font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Cultivate the Bacteria</b></font></h3><br />
<font face="Arial, Helvetica"><p>According to the results of the PCR detection, we choose positive colonies and transfer them to 5ml LB liquid media ( 5μl of ampicillin has added) stored in 12.5ml centrifuge tubes. Put the centrifuge tubes in 37℃ gas bath overnight. </p><br />
<p><font face="Arial, Helvetica"><a name="Plasmid_DNA"></a> </font></p><br />
<font face="Arial, Helvetica"> </font> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Plasmid DNA Isolation</b></font></h3><br />
<p><font face="Arial, Helvetica">Use E.Z.N.A.TM Plasmid Mini I to isolate the constructed plasmid mutant-psb1a3-GR. </font></p><br />
<p><font face="Arial, Helvetica"><a name="Restriction_Enzyme" ></a> </font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Restriction Enzyme Digestion and Electrophoresis</b></font></h3><br />
<p>From the last step, we got the certain quantities of isolated plasmids. In this step, we do two restriction enzyme digestion reactions, one to prove that the plasmid is construct correctly ( mutant-psb1a3-GR ), one to get sticky ends preparing for the ligation.<br /><br />
<strong>Method</strong></p><br />
<ul><br />
<li><strong>Restriction Enzyme Digestion to prove that plasmid is constructed correctly</strong></li><br />
</ul><br />
<p align="left">a. Prepare the sample reaction as indicated below:<br /><br />
Total: 10μl<br /><br />
+ 1.0μl of Not I restriction enzyme,#ER0591<br /><br />
+ 1.0μl of Spe I restriction enzyme,#ER1251 <br /><br />
+ 2.0μl of Buffer Tango( 10X )<br /><br />
+ 1.5μl of plasmid mutant-psb1a3-GR<br /><br />
+ 14.5μl of ddH2O <br /><br />
b. Electrophorese the total system and observe the lane separation. </p><br />
<ul><br />
<li><strong>Restriction Enzyme Digestion to get sticky ends preparing for the ligation.</strong></li><br />
</ul><br />
<p align="left">a. Prepare the sample reaction as indicated below:<br /><br />
Total: 50μl<br /><br />
+ 5.0μl of Pst I restriction enzyme, #ER0611<br /><br />
+ 5.0μl of Xba I restriction enzyme, #ER0681 <br /><br />
+ 3.0μl of Buffer Tango( 10X )<br /><br />
+ 5.0μl of plasmid mutant-psb1a3-GR<br /><br />
+ 32.0μl of ddH2O <br /><br />
b. Electrophorese the total system and observe the lane separation.<br /><br />
c. Cut the gel of specific position and collect it in tubes that have measured weight. <br /><br />
d. Use DNA Gel Extraction Ki to purify the plasmid DNA mutant-psb1a3-GR(PX) .<br /><br />
Note: Mutant-psb1a3-GR(PX) means plasmid Mutant-psb1a3-GR digested by Pst I and Xba I.</p><br />
<p><strong>Figure</strong></p><br />
<p align="left"><strong><img src="https://static.igem.org/mediawiki/2012/d/d8/11111.png" alt="" class="img_fl img_border" /></strong></p><br />
<p>(Figure 4 : The double digestion of mutant-psb1a3 forms a linear DNA fragments and it runs slower than circle DNA fragments. This suggest that the double digestion of mutant-psb1a3 works in a high efficiency, and desired sticky ends are formed.1,3,5 are plasmids digested by restriction enzyme Pst I and Xba I from different colonies, 2,5,6 are pure plasmid mutant-psb1a3-GR, 7 is the plasmid mutant-psb1a3. )</p><br />
<p align="left">&nbsp;</p><br />
<p><font face="Arial, Helvetica"><a name="Ligation1" ></a> </font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Ligation</b></font></h3><br />
<p>Ligation is the process that target DNA gene is inserted into a plasmid. Both the vector and insert are prepared to have the sticky ends. These two kinds of DNA pieces are placed in a reaction tube and the proper DNA ligase, buffer, and cofactors are added for the reaction to take place. When done properly, the ligation will result in a successful combination of the insert and plasmid into one plasmid.Based on the digestion of mutant-psb1a3-GR, terminator DNA fragments,ligation can be done. We ligate mutant-psb1a3-GR vector and sticky terminator DNA fragments to construct an new plasmid mutant-psb1a3-GR-t.By detecting the quantities of GFP and RFP, terminator efficiency can be calculated. <br /><br />
1. Prepare the control reaction as indicated below:<br /><br />
Total: 10μl <br /><br />
+ 1.0μl of plasmid mutant-psb1a3 (NA) <br /><br />
+ 6.0μl of terminator<br /><br />
+ 2.0μl of T4 DNA Ligase , #EL0011 <br /><br />
+ 1.0μl of 10XT4 Ligase buffer<br /><br />
2. Put the tubes in 22℃ water bath, react for 8-12 hours. </p><br />
<p><font face="Arial, Helvetica"><a name="Bacteria" id="Culture_the"></a> </font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Bacteria transformation</b></font></h3><br />
<p>Transform the ligation products into the DH-5α competent cells, put the plate on 37℃ gas bath for 12-16 hours. </p><br />
<p><font face="Arial, Helvetica"><a name="Cultivate"></a> </font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Cultivate the Bacteria</b></font></h3><br />
<p>According to the results of the PCR detection, we choose positive colonies and transfer them to 5ml LB liquid media ( 5μl of ampicillin has added) stored in 12.5ml centrifuge tubes. Put the centrifuge tubes in 37℃ gas bath overnight. </p><br />
<p><font face="Arial, Helvetica"><a name="Flow" ></a> </font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Flow Cytometer Analysis</b></font></h3><br />
<p> 1. NaCl solution( 0.9% )<br /><br />
2. 75% Methanol<br /><br />
B. Procedures:<br /><br />
1. Transfer overnight suspension culture to 1.5ml centrifuge tubes and centrifuge at 12000RPM for 30s. Pour the supernatant and add overnight suspension culture and centrifuge again until enough bacteria has been collected. <br /><br />
2. Use NaCl solution(0.9%) to mix the bacteria and vibrate the centrifuge tubes until the bacteria distributed uniformly.<br /><br />
3. Put the centrifuge tubes into the Flow Cytometer and set parameters and run the program.</p><br />
<p>&nbsp;</p><br />
<p><font face="Arial, Helvetica"><a name="Fluorescence"></a> </font></p><br />
<font face="Arial, Helvetica"> </font><br />
<h3><font color="#0000FF" face="Arial, Helvetica"><b>Fluorescence Microscope</b></font></h3><br />
<p><strong>Method</strong><br /><br />
Add 10μl of former bacteria solution to micro slide and cover with coverslip.Then placed it on the Fluorescence Microscope with 488nm light activating and observe the GFP and RFP. </p><br />
<br> <br />
<br />
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</html></div>M.B.ZHOUhttp://2012.igem.org/Team:SUSTC-Shenzhen-A/week13Team:SUSTC-Shenzhen-A/week132012-10-26T17:23:07Z<p>M.B.ZHOU: </p>
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<h1 class="title">Week13~Week18</h1><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;</p><br />
<ul><br />
<br />
<li>Week13</li><br />
</ul><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;</p><br />
<br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;Asia Jamboree in HKUST!</p><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;We met many teams from China, Japan, Korea, India and Indoneisa. We presented a high quality presentation. Yet we received many good advices from the judges. And we showed our poster and made many friends! But it is a little upset that we only got a silver medal. We think that we deserved a gold medal. Because we think we fulfilled every requirements of a gold medal. </p><br />
<br/><br/><br />
<ul><br />
<li>Week 14, 15</li><br />
</ul><br />
<br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;First two weeks from Hong Kong. We were a little upset since we were not invited to MIT. So we did nothing these two weeks. </p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><p>&nbsp;&nbsp;</p><br />
<br />
<ul><br />
<li>Week 16</li><br />
</ul><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;Good news! We were invited to MIT, too! We came back to our second project---BioDesign. Xiao Tong and Zili Fan were in charge of the drawing part. They spent virtually all the spare time on this project. Sometimes they even skipped classes to write code. Mubing Zhou was in charge of the rest part other than the drawing part. He was also very hardworking. Deng Pan and Yujun Zhao were in charge of updating BioSearch. While Qijia Cheng, Chenchen Lyu and Junqiu Zhang were busy preparing the presentation. Yidan Pan and Xin Yang were in charge of the wiki writing. And Jingyao Guo and Yiqi Jiang were in charge of name card making and poster redesigning.</p><br />
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<p class="title1">Notebook</p><br />
<img src="https://static.igem.org/mediawiki/2012/c/c9/Devidingline_side.jpg"><br />
<p><a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/Notebook"><strong>Overview</strong></a></p><br />
<p><strong>Preparation</strong></p><br />
<p><a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/JC"><strong>Journal Club</strong></a></p><br />
<p><a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/FP"><strong>Final Project</strong></a></p><br />
<p><a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/week1"><strong>Xcode Tutorial(week 1)</strong></a></p><br />
<p><strong>Biosearch section1(week2,3)</strong></p><br />
<p>&nbsp;&nbsp;<a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/week2">week 2</a></p><br />
<p>&nbsp;&nbsp;<a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/week3">week 3</a></p><br />
<p><strong>Biosearch section2(week4,5)</strong></p><br />
<p><a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/week4&5"><strong>week4~5</strong></a></p><br />
<p><strong>Tinker Cell(week,6,7)</strong></p><br />
<p>&nbsp;&nbsp;<a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/week6">week 6</a></p><br />
<p>&nbsp;&nbsp;<a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/week7">week 7</a></p><br />
<p><strong>Comprehensive work</strong></p><br />
<p>&nbsp;&nbsp;<a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/week8">week 8</a></p><br />
<p>&nbsp;&nbsp;<a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/week9">week 9~12</a></p><br />
<p>&nbsp;&nbsp;<a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/week13">week 13~18</a></p><br />
<br />
<div class="sidebar_box_bottom"></div> <br />
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<ul><br />
<li>Week 17</li><br />
</ul><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;Since we had had some experience in writing codes, we didn't discuss too much when developing BioDesign. This week. We kept working hard. Xiao Tong, Zili Fan and Mubing Zhou already finished BioDesign. But there was a huge task remaining--debugging. Hundreds of bugs were hidden between lines. Sometimes, they spent half a day just to fix a tiny problem. Other guys were still doing their jobs. Jingyao Guo and Yiqi Jiang printed our name cards and new poster. Deng Pan and Yujun Zhao fixed some bugs in BioSearch. </p><br />
<br />
<td/><tr/><br />
<br />
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<ul><br />
<li>Week 18</li><br />
</ul><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;World Jamboree in MIT! </p><br />
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[[file:footbar.jpg|center]]</div>M.B.ZHOUhttp://2012.igem.org/Team:SUSTC-Shenzhen-A/Biodesign_TutorialTeam:SUSTC-Shenzhen-A/Biodesign Tutorial2012-10-26T16:24:50Z<p>M.B.ZHOU: </p>
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<h1 class="title">&nbsp;BioDesign Tutorial</h1>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<ul><br />
<a href="#Folder">Folder page <br />
</a> &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
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<a href="#File">File page <br />
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<a href="#Drawing">Drawing page <br />
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<a href="#Mail">Mail page <br />
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<p class="title1">&nbsp;&nbsp;Folder page</p><br />
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<p><img src="https://static.igem.org/mediawiki/2012/e/e6/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_1.png" valign="top" align="left" width="300" height = "480" style="BORDER:#CCFFCC 5px dashed;margin:10px;" ><img src="https://static.igem.org/mediawiki/2012/b/b7/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_2.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:10px;" ></p><br />
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<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(1)</p><br />
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<p>&nbsp;&nbsp;Fig.1 is the first page. </p><br />
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<p>&nbsp;&nbsp;In order to create a new project/folder, you should click the ‘add’ button at the top right (Fig.2).</p><br />
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<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(2)--></p><br />
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<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(3)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the icon, then click ‘enter’,<br/> it goes into the secondary page (Fig.3).</p><br />
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<p class="title1">&nbsp;&nbsp;File page</p><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/Devidingline_whole.jpg"/><br />
<p><img src="https://static.igem.org/mediawiki/2012/2/26/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_4.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/6/60/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_5.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:10px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(4)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;The secondary page looks like Fig.4. </p><br />
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<p>&nbsp;&nbsp;</p><br />
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<p>&nbsp;&nbsp;Click the button name ‘add’ at the top right to create a new file (Fig.5).</p><br />
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<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(5)--></p><br />
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<p class="title1">&nbsp;&nbsp;Drawing page</p><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/Devidingline_whole.jpg"/><br />
<p><img src="https://static.igem.org/mediawiki/2012/4/41/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_6.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/5/54/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_7.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(6)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the newly created file, choose ‘edit’ to do the drawing (Fig.6).</p><br />
<p>&nbsp;&nbsp;</p><br />
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<p>&nbsp;&nbsp;Click a object at the bottom to set it on the screen.</p><br />
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<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(7)--></p><br />
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<p><img src="https://static.igem.org/mediawiki/2012/8/8f/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_8.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/b/b2/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_9.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(8)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the text field next to a part to rename this part.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;For the objects in “comp”, you can zoom them by ywo fingers to change the their sizes.</p><br />
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<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(9)--></p><br />
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<p><img src="https://static.igem.org/mediawiki/2012/1/14/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_10.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/f/f6/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_11.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(10)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can rotate the object clockwisely by clicking the rotating button.</p><br />
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<p>&nbsp;&nbsp;You can get a vector on the canvas from “part”.</p><br />
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<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(11)--></p><br />
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<p><img src="https://static.igem.org/mediawiki/2012/f/fd/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_12.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/c/c8/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_13.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(12)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can change the size of the vector by zooming with two fingers.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;If you have add a cell, you may find it covers the vector or other object. Click the “down” button to lower layer.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(13)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/6/63/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_14.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/7/71/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_15.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(14)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can put objects(in”mole. & part.”) on vector by draging it into the vector.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;In “reac.”, you have various choices to connect objects by Bezier curve.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(15)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/2/24/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_16.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/f/fa/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_17.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(16)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click two objects in turn and they will be connected.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can click the place near the line to pitch the curve. And then change the shape of the curve by dragging with one or two fingers.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(17)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/c/cb/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_18.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/9/99/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_19.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(18)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can change the type of the arrow by clicking the first button at the right.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Fig.19 shows the changed arrow.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(19)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/7/77/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_20.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/0/0e/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_21.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(20)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can pitch the center point to edit both parts at the same time.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;In “regu.”, choose one to connect two or three parts by polygonal line.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(21)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/c/ca/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_22.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/0/00/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_23.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(22)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Fig.22 shows what happens after choosing the "regu".</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Fig.23 also shows what happens after choosing the "regu".</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(23)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/6/66/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_24.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/6/6d/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_25.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(24)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;For objects in “part.”, you can move one group close to another to connet them.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can press the button to disconnect them.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(25)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/d/df/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_70.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/9/90/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_71.png" valign="top" align="right" width="300"height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(26)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the button at the top right to save this picture (Fig.26). </p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Come back, you’ll see the icon has change into the saved picture (Fig.27).</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(27)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/e/ef/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_72.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/f/f8/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_73.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(28)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the microscope button in FIle page at the bottom to search a file (Fig.28). </p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbspClick the button at the bottom right to present the files in a table (Fig.29).</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(29)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><a name="Mail" ></a> <br />
<p class="title1">&nbsp;&nbsp;Mail page</p><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/Devidingline_whole.jpg" /><br />
<p><img src="https://static.igem.org/mediawiki/2012/b/bf/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_74.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/5/5a/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_75.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:10px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(30)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Come back to the Folder page. Click the envelope button at the bottom right to the file that you want to email to your friend! (Fig.30)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the ‘done’ button. Send the chosen files to your friends (Fig.31)!</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(31)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><a name="Video" ></a> <br />
<p class="title1">&nbsp;&nbsp;Video tutorial</p><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/Devidingline_whole.jpg" /><br />
<p>&nbsp;&nbsp;</p><br />
<p align="center"><br />
<embed src="http://player.youku.com/player.php/sid/XNDY3MDg5NTk2/v.swf" <br />
width="480" height="400" <br />
type="application/x-shockwave-flash"><br />
</embed></p><br />
<br />
</td><br />
</tr><br />
<br />
</table><br />
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[[file:footbar.jpg|center]]</div>M.B.ZHOUhttp://2012.igem.org/Team:SUSTC-Shenzhen-A/Biodesign_TutorialTeam:SUSTC-Shenzhen-A/Biodesign Tutorial2012-10-26T16:10:04Z<p>M.B.ZHOU: </p>
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<td><div><br />
<h1 class="title">&nbsp;BioDesign Tutorial</h1>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<ul><br />
<a href="#Folder">Folder page <br />
</a> &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
<br />
<a href="#File">File page <br />
</a>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
<br />
<a href="#Drawing">Drawing page <br />
</a>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
<br />
<a href="#Mail">Mail page <br />
</a> &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
<a href="#Video">Video tutorial<br />
</a> <br />
</ul><br />
<table border="0" cellspacing="0" cellpadding="0"><br />
<tr><br />
<td>&nbsp;</td><br />
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<br />
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<tr><td><a name="Folder" ></a> <br />
<p class="title1">&nbsp;&nbsp;Folder page</p><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/Devidingline_whole.jpg"><br />
<br />
<p><img src="https://static.igem.org/mediawiki/2012/e/e6/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_1.png" valign="top" align="left" width="300" height = "480" style="BORDER:#CCFFCC 5px dashed;margin:10px;" ><img src="https://static.igem.org/mediawiki/2012/b/b7/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_2.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:10px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(1)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Fig.1 is the first page. </p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;In order to create a new project/folder, you should click the ‘add’ button at the top right (Fig.2).</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(2)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/3/35/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_3.png" valign="top" align="left" height="480" width="300" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(3)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the icon, then click ‘enter’,<br/> it goes into the secondary page (Fig.3).</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><a name="File" ></a> <br />
<p class="title1">&nbsp;&nbsp;File page</p><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/Devidingline_whole.jpg"><br />
<p><img src="https://static.igem.org/mediawiki/2012/2/26/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_4.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/6/60/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_5.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:10px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(4)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;The secondary page looks like Fig.4. </p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the button name ‘add’ at the top right to create a new file (Fig.5).</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(5)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<a name="Drawing"></a><br />
<p class="title1">&nbsp;&nbsp;Drawing page</p><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/Devidingline_whole.jpg"><br />
<p><img src="https://static.igem.org/mediawiki/2012/4/41/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_6.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/5/54/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_7.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(6)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the newly created file, choose ‘edit’ to do the drawing (Fig.6).</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click a object at the bottom to set it on the screen.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(7)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/8/8f/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_8.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/b/b2/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_9.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(8)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the text field next to a part to rename this part.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;For the objects in “comp”, you can zoom them by ywo fingers to change the their sizes.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(9)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/1/14/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_10.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/f/f6/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_11.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(10)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can rotate the object clockwisely by clicking the rotating button.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can get a vector on the canvas from “part”.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(11)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/f/fd/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_12.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/c/c8/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_13.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(12)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can change the size of the vector by zooming with two fingers.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;If you have add a cell, you may find it covers the vector or other object. Click the “down” button to lower layer.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(13)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/6/63/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_14.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/7/71/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_15.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(14)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can put objects(in”mole. & part.”) on vector by draging it into the vector.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;In “reac.”, you have various choices to connect objects by Bezier curve.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(15)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/2/24/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_16.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/f/fa/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_17.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(16)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click two objects in turn and they will be connected.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can click the place near the line to pitch the curve. And then change the shape of the curve by dragging with one or two fingers.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(17)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/c/cb/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_18.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/9/99/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_19.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(18)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can change the type of the arrow by clicking the first button at the right.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Fig.19 shows the changed arrow.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(19)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/7/77/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_20.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/0/0e/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_21.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(20)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can pitch the center point to edit both parts at the same time.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;In “regu.”, choose one to connect two or three parts by polygonal line.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(21)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/c/ca/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_22.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/0/00/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_23.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(22)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Fig.22 shows what happens after choosing the "regu".</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Fig.23 also shows what happens after choosing the "regu".</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(23)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/6/66/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_24.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/6/6d/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_25.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(24)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;For objects in “part.”, you can move one group close to another to connet them.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can press the button to disconnect them.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(25)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/d/df/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_70.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/9/90/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_71.png" valign="top" align="right" width="300"height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(26)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the button at the top right to save this picture (Fig.26). </p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Come back, you’ll see the icon has change into the saved picture (Fig.27).</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(27)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/e/ef/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_72.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/f/f8/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_73.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(28)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the microscope button in FIle page at the bottom to search a file (Fig.28). </p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbspClick the button at the bottom right to present the files in a table (Fig.29).</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(29)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><a name="Mail" ></a> <br />
<p class="title1">&nbsp;&nbsp;Mail page</p><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/Devidingline_whole.jpg"><br />
<p><img src="https://static.igem.org/mediawiki/2012/b/bf/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_74.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/5/5a/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_75.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:10px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(30)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Come back to the Folder page. Click the envelope button at the bottom right to the file that you want to email to your friend! (Fig.30)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the ‘done’ button. Send the chosen files to your friends (Fig.31)!</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(31)--></p><br />
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<p class="title1">&nbsp;&nbsp;Video tutorial</p><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/Devidingline_whole.jpg"><br />
<p>&nbsp;&nbsp;</p><br />
<p align="center"><embed src="http://player.youku.com/player.php/sid/XNDY3MDg5NTk2/v.swf" play="true" quality="high" width="540" height="400" align="middle" allowScriptAccess="always" type="application/x-shockwave-flash"></embed></p><a name="video"></a><br />
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[[file:footbar.jpg|center]]</div>M.B.ZHOUhttp://2012.igem.org/Team:SUSTC-Shenzhen-A/RFCTeam:SUSTC-Shenzhen-A/RFC2012-10-26T14:50:30Z<p>M.B.ZHOU: </p>
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<h1 class="title"><strong>Introduction</strong></h1><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;In 2003, the first attempt to make a synthetic biology a discipline that everyone can share about it was made. After the appearance of the first BioBrick(tm), the concept about standardization had been widely accepted. And it is a trend in synthetic biology now. </p><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;Thanks to the standardization, biologists have developed many useful tools with the help of the standardized parts, the BioBricks. These BioBricks really contribute a lot to the research field. And they make it easier for researchers to communicate with each other under the same technical standards. Due to the convenience of BioBricks, thousands of BioBricks have been created and introduced during these years. Thus the database containing these BioBricks is getting larger and larger.</p><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;But here comes the problem. Since every one can share their BioBricks, sometimes the information of the BioBricks are incomplet when they submit. So it becomes hard for users to search for a BioBrick that they want. Although there is an RFC, RFC 52 already, but we are not satisfied. Because the description of it is too general, the database of the BioBricks is still a mess. So we want to create a new RFC to replace RFC 52.</p><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;We want to establish an RFC to describe the minimum information for a qualified BioBrick. We listed the minimum information a BioBrick should have, and gave a short description for each of them. Especially, we want to qualify the classification of BioBricks. All the BioBricks should be organized, so that users can search for it easily.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<h1 class="title"><strong>The current problem</strong></h1><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title1">&nbsp;&nbsp;Information is not complete in partsregistry.org</p><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;When the <a href="http://partsregistry.org/Main_Page">partsregistry.org</a> was established at first, it is worth to be called the authority of the BioBricks. But as time goes by, more and more BioBricks are added in, now <a href="http://partsregistry.org/Main_Page">partsregistry.org</a> becomes less satisfy. Information of many BioBricks are incomplete, some even wrong. Because every one can submit one's BioBrick as long as one logs in, many information is informal since the website doesn't examine the information users submit. This would waste resource as well as mess up the database.</p><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;Moreover, the layout of the website becomes more unclear as the database becomes larger and larger. For example, the Catalog. In this page, BioBricks are classified according to many different criteria. But the interface style in one section is far different from it in another section. Sometimes it is the description of a project, sometimes it becomes BioBrick list. A large list will be prsented in front of users with hundreds of BioBricks, but no search tool is available for users to search for the BioBrick that they really want. All of these would increase the difficulty for users to find the information that they want. For example, in the following figure, no list for Translational units can be found while others have long lists.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p align="center">&nbsp;&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2012/b/b5/Problem1.jpg"></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>This BioBrick is totally useless but it is still on the BioBrick list. This shows that the uploading of BioBricks should be standardized.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p align="center">&nbsp;&nbsp;&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2012/e/e0/Problem2.jpg"></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title1">&nbsp;&nbsp;Information is not useful in database</p><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;We got a database that contains all the information about all the BioBricks. In this database, every BioBrick has 40 properties, they are:</p><br />
<p>&nbsp;&nbsp;</p><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2012/6/6d/Properties_list.png"></p><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;With these long list, nobody has the patience to look through them all. Indeed many properties are rubbish. Look at the property No.34-No.37, they are completely useless because they are apparently not a part of information of a BioBrick. Another example is property No.14, specified_scar_u_list. This property is NULL, which means it contains no information, for nearly all the BioBricks. We draw a histogram to demonstrate the percentage of unqualified and qualified BioBricks.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p align="center">&nbsp;&nbsp;<img src="https://static.igem.org/mediawiki/2012/2/22/Histogram_for_database.png"></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;Explanation of the above histogram:</p><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;x-axis:&nbsp;&nbsp;&nbsp; 1 - number o properties whose information is NULL for more 3000 BioBricks (our database contains 13442 BioBricks).</p> <p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2 - number of properties whose information for most BioBrick is a number '0'.</p><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;3 - number of properties whose information for most BioBrick is some random number.</p><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;4 - number of properties with valid information.</p><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;y-axis:&nbsp;&nbsp;&nbsp; number of corresponding properties</p><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;We can conclude from the above histogram that only one fourth of the properties are valid properties. So most information in the database is trash. Because of the mess in the database, it is essential to standardize the minimum information for a qualified BioBrick.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title"><strong>Our BBF RFC 89:</strong></p><h1 class="title"><strong>The minimum information for a qualified BioBrick</strong></h1><br />
<h3>Motivation to write this BBF RFC</h3><br />
<p>&nbsp;&nbsp;Since the information of many existing BioBricks is incomplete, thus the usage of the BioBricks will be affected. It is necessary to standardize the minimum information required for a qualified BioBrick. Furthermore this standardization will reduce the time to locate and find a BioBrick. So it is essential to create a default template for storing the information of BioBricks.</p><br />
<br/><br />
<p><big>&nbsp;&nbsp;To see the complete version of this BBF RFC, please click <a href="https://2012.igem.org/Team:SUSTC-Shenzhen-A/RFC_draft">here!</a></big></p><br />
<br/><br />
<p><big>&nbsp;&nbsp;To download this BBF RFC, please click <a href="http://dspace.mit.edu/handle/1721.1/73910">here!</a></big></p><br />
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[[file:footbar.jpg|center]]</div>M.B.ZHOUhttp://2012.igem.org/Team:SUSTC-Shenzhen-A/Biodesign_TutorialTeam:SUSTC-Shenzhen-A/Biodesign Tutorial2012-10-26T14:43:52Z<p>M.B.ZHOU: </p>
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<h1 class="title">&nbsp;BioDesign Tutorial</h1>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<ul><br />
<a href="#Folder">Folder page <br />
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<p class="title1">&nbsp;&nbsp;Folder page</p><br />
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<p><img src="https://static.igem.org/mediawiki/2012/e/e6/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_1.png" valign="top" align="left" width="300" height = "480" style="BORDER:#CCFFCC 5px dashed;margin:10px;" ><img src="https://static.igem.org/mediawiki/2012/b/b7/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_2.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:10px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(1)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Fig.1 is the first page. </p><br />
<p>&nbsp;&nbsp;</p><br />
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<p>&nbsp;&nbsp;</p><br />
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<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;In order to create a new project/folder, you should click the ‘add’ button at the top right (Fig.2).</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(2)--></p><br />
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<p><img src="https://static.igem.org/mediawiki/2012/3/35/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_3.png" valign="top" align="left" height="480" width="300" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(3)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the icon, then click ‘enter’,<br/> it goes into the secondary page (Fig.3).</p><br />
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<p class="title1">&nbsp;&nbsp;File page</p><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/Devidingline_whole.jpg"><br />
<p><img src="https://static.igem.org/mediawiki/2012/2/26/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_4.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/6/60/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_5.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:10px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(4)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;The secondary page looks like Fig.4. </p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the button name ‘add’ at the top right to create a new file (Fig.5).</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(5)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<a name="Drawing"></a><br />
<p class="title1">&nbsp;&nbsp;Drawing page</p><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/Devidingline_whole.jpg"><br />
<p><img src="https://static.igem.org/mediawiki/2012/4/41/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_6.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/5/54/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_7.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(6)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the newly created file, choose ‘edit’ to do the drawing (Fig.6).</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click a object at the bottom to set it on the screen.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(7)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/8/8f/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_8.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/b/b2/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_9.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(8)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the text field next to a part to rename this part.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;For the objects in “comp”, you can zoom them by ywo fingers to change the their sizes.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(9)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/1/14/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_10.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/f/f6/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_11.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(10)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can rotate the object clockwisely by clicking the rotating button.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can get a vector on the canvas from “part”.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(11)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/f/fd/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_12.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/c/c8/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_13.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(12)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can change the size of the vector by zooming with two fingers.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;If you have add a cell, you may find it covers the vector or other object. Click the “down” button to lower layer.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(13)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/6/63/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_14.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/7/71/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_15.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(14)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can put objects(in”mole. & part.”) on vector by draging it into the vector.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;In “reac.”, you have various choices to connect objects by Bezier curve.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(15)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/2/24/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_16.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/f/fa/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_17.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(16)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click two objects in turn and they will be connected.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can click the place near the line to pitch the curve. And then change the shape of the curve by dragging with one or two fingers.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(17)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/c/cb/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_18.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/9/99/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_19.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(18)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can change the type of the arrow by clicking the first button at the right.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Fig.19 shows the changed arrow.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(19)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/7/77/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_20.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/0/0e/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_21.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(20)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can pitch the center point to edit both parts at the same time.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;In “regu.”, choose one to connect two or three parts by polygonal line.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(21)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/c/ca/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_22.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/0/00/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_23.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(22)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Fig.22 shows what happens after choosing the "regu".</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Fig.23 also shows what happens after choosing the "regu".</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(23)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/6/66/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_24.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/6/6d/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_25.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(24)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;For objects in “part.”, you can move one group close to another to connet them.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can press the button to disconnect them.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(25)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/d/df/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_70.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/9/90/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_71.png" valign="top" align="right" width="300"height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(26)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the button at the top right to save this picture (Fig.26). </p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Come back, you’ll see the icon has change into the saved picture (Fig.27).</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(27)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/e/ef/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_72.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/f/f8/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_73.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(28)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the microscope button in FIle page at the bottom to search a file (Fig.28). </p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbspClick the button at the bottom right to present the files in a table (Fig.29).</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(29)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><a name="Mail" ></a> <br />
<p class="title1">&nbsp;&nbsp;Mail page</p><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/Devidingline_whole.jpg"><br />
<p><img src="https://static.igem.org/mediawiki/2012/b/bf/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_74.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/5/5a/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_75.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:10px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(30)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Come back to the Folder page. Click the envelope button at the bottom right to the file that you want to email to your friend! (Fig.30)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the ‘done’ button. Send the chosen files to your friends (Fig.31)!</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(31)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><a name="Video" ></a> <br />
<p class="title1">&nbsp;&nbsp;Video tutorial</p><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/Devidingline_whole.jpg"><br />
<br />
</td><br />
</tr><br />
<br />
</table><br />
</p><br />
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[[file:footbar.jpg|center]]</div>M.B.ZHOUhttp://2012.igem.org/File:SUSTC-Shenzhen-A_BioDesign_tutorial_fig_25.pngFile:SUSTC-Shenzhen-A BioDesign tutorial fig 25.png2012-10-26T14:38:40Z<p>M.B.ZHOU: </p>
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<div></div>M.B.ZHOUhttp://2012.igem.org/File:SUSTC-Shenzhen-A_BioDesign_tutorial_fig_24.pngFile:SUSTC-Shenzhen-A BioDesign tutorial fig 24.png2012-10-26T14:37:06Z<p>M.B.ZHOU: uploaded a new version of &quot;File:SUSTC-Shenzhen-A BioDesign tutorial fig 24.png&quot;</p>
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<div></div>M.B.ZHOUhttp://2012.igem.org/File:SUSTC-Shenzhen-A_BioDesign_tutorial_fig_24.pngFile:SUSTC-Shenzhen-A BioDesign tutorial fig 24.png2012-10-26T14:35:47Z<p>M.B.ZHOU: </p>
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<div></div>M.B.ZHOUhttp://2012.igem.org/Team:SUSTC-Shenzhen-A/Biodesign_TutorialTeam:SUSTC-Shenzhen-A/Biodesign Tutorial2012-10-26T14:33:31Z<p>M.B.ZHOU: </p>
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<div> <tr><br />
<td><div><br />
<h1 class="title">&nbsp;BioDesign Tutorial</h1>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<ul><br />
<a href="#Folder">Folder page <br />
</a> &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
<br />
<a href="#File">File page <br />
</a>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
<br />
<a href="#Drawing">Drawing page <br />
</a>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
<br />
<a href="#Mail">Mail page <br />
</a> &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
<a href="#Video">Video tutorial<br />
</a> <br />
</ul><br />
<table border="0" cellspacing="0" cellpadding="0"><br />
<tr><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<br />
<tr><td><a name="Folder" ></a> <br />
<p class="title1">&nbsp;&nbsp;Folder page</p><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/Devidingline_whole.jpg"><br />
<br />
<p><img src="https://static.igem.org/mediawiki/2012/e/e6/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_1.png" valign="top" align="left" width="300" height = "480" style="BORDER:#CCFFCC 5px dashed;margin:10px;" ><img src="https://static.igem.org/mediawiki/2012/b/b7/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_2.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:10px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(1)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Fig.1 is the first page. </p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;In order to create a new project/folder, you should click the ‘add’ button at the top right (Fig.2).</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(2)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
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<p><img src="https://static.igem.org/mediawiki/2012/3/35/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_3.png" valign="top" align="left" height="480" width="300" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(3)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the icon, then click ‘enter’,<br/> it goes into the secondary page (Fig.3).</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
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</td><br />
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<tr><br />
<td><a name="File" ></a> <br />
<p class="title1">&nbsp;&nbsp;File page</p><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/Devidingline_whole.jpg"><br />
<p><img src="https://static.igem.org/mediawiki/2012/2/26/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_4.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/6/60/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_5.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:10px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(4)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;The secondary page looks like Fig.4. </p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the button name ‘add’ at the top right to create a new file (Fig.5).</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(5)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
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</td><br />
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<td><br />
<a name="Drawing"></a><br />
<p class="title1">&nbsp;&nbsp;Drawing page</p><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/Devidingline_whole.jpg"><br />
<p><img src="https://static.igem.org/mediawiki/2012/4/41/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_6.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/5/54/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_7.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(6)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the newly created file, choose ‘edit’ to do the drawing (Fig.6).</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click a object at the bottom to set it on the screen.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(7)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
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</td><br />
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<br />
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<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/8/8f/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_8.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/b/b2/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_9.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(8)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the text field next to a part to rename this part.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;For the objects in “comp”, you can zoom them by ywo fingers to change the their sizes.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(9)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/1/14/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_10.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/f/f6/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_11.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(10)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can rotate the object clockwisely by clicking the rotating button.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can get a vector on the canvas from “part”.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(11)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
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<p>&nbsp;&nbsp;</p><br />
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</td><br />
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<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/f/fd/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_12.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/c/c8/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_13.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(12)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can change the size of the vector by zooming with two fingers.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;If you have add a cell, you may find it covers the vector or other object. Click the “down” button to lower layer.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(13)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
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</td><br />
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<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/6/63/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_14.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/7/71/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_15.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(14)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can put objects(in”mole. & part.”) on vector by draging it into the vector.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;In “reac.”, you have various choices to connect objects by Bezier curve.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(15)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
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<br/><br />
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</td><br />
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<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/2/24/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_16.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/f/fa/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_17.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(16)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click two objects in turn and they will be connected.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can click the place near the line to pitch the curve. And then change the shape of the curve by dragging with one or two fingers.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(17)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
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<br/><br />
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<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/c/cb/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_18.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/9/99/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_19.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(18)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can change the type of the arrow by clicking the first button at the right.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Fig.19 shows the changed arrow.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(19)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
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<p><img src="https://static.igem.org/mediawiki/2012/7/77/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_20.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/0/0e/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_21.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(20)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can pitch the center point to edit both parts at the same time.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;In “regu.”, choose one to connect two or three parts by polygonal line.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(21)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/c/ca/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_22.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/0/00/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_23.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(22)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Fig.22 shows what happens after choosing the "regu".</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Fig.23 also shows what happens after choosing the "regu".</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(23)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/d/df/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_70.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/9/90/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_71.png" valign="top" align="right" width="300"height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(70)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the button at the top right to save this picture (Fig.70). </p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Come back, you’ll see the icon has change into the saved picture (Fig.71).</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(71)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/e/ef/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_72.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/f/f8/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_73.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(72)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the microscope button in FIle page at the bottom to search a file (Fig.72). </p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbspClick the button at the bottom right to present the files in a table (Fig.73).</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(73)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><a name="Mail" ></a> <br />
<p class="title1">&nbsp;&nbsp;Mail page</p><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/Devidingline_whole.jpg"><br />
<p><img src="https://static.igem.org/mediawiki/2012/b/bf/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_74.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/5/5a/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_75.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:10px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(74)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Come back to the Folder page. Click the envelope button at the bottom right to the file that you want to email to your friend! (Fig.74)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the ‘done’ button. Send the chosen files to your friends (Fig.75)!</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(75)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><a name="More" ></a> <br />
<p class="title1">&nbsp;&nbsp;More</p><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/Devidingline_whole.jpg"><br />
<p><img src="https://static.igem.org/mediawiki/2012/d/dc/BiosearchPage16.png" valign="top" align="left" width="300" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/6/64/BiosearchPage17.png" valign="top" align="right" width="300" style="BORDER:#CCFFCC 5px dashed;margin:10px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(16)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;In the "more" page, you can get some information about synthetic biology and biobricks.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;On the bottom, there is the feedback part.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(17)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/e/ef/BiosearchPage19.png" valign="top" align="left" width="300" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/5/5c/BiosearchPage20.png" valign="top" align="right" width="300" style="BORDER:#CCFFCC 5px dashed;margin:10px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(18)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;The ‘About us’ part show the information of our team.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the ‘Compose the mail’ to send your suggestion to us.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(19)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</br><br />
</br><br />
</br><br />
</br><br />
<a name="Video"></a><br />
<p class="title1">&nbsp;&nbsp;Simplified Video tutorial</p><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/Devidingline_whole.jpg"><br />
<p>&nbsp;&nbsp;</p><br />
<p align="center"><embed src="http://player.youku.com/player.php/sid/XNDY3MDg5NTk2/v.swf" play="true" quality="high" width="540" height="400" align="middle" allowScriptAccess="always" type="application/x-shockwave-flash"></embed></p><a name="video"></a><br />
</td><br />
</tr><br />
<br />
</table><br />
</p><br />
<div></td><br />
</tr><br />
</table><br />
</table><br />
<br />
<br />
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</html><br />
[[file:footbar.jpg|center]]</div>M.B.ZHOUhttp://2012.igem.org/File:SUSTC-Shenzhen-A_BioDesign_tutorial_fig_23.pngFile:SUSTC-Shenzhen-A BioDesign tutorial fig 23.png2012-10-26T14:31:23Z<p>M.B.ZHOU: </p>
<hr />
<div></div>M.B.ZHOUhttp://2012.igem.org/File:SUSTC-Shenzhen-A_BioDesign_tutorial_fig_22.pngFile:SUSTC-Shenzhen-A BioDesign tutorial fig 22.png2012-10-26T14:31:02Z<p>M.B.ZHOU: </p>
<hr />
<div></div>M.B.ZHOUhttp://2012.igem.org/File:SUSTC-Shenzhen-A_BioDesign_tutorial_fig_21.pngFile:SUSTC-Shenzhen-A BioDesign tutorial fig 21.png2012-10-26T14:27:55Z<p>M.B.ZHOU: </p>
<hr />
<div></div>M.B.ZHOUhttp://2012.igem.org/File:SUSTC-Shenzhen-A_BioDesign_tutorial_fig_20.pngFile:SUSTC-Shenzhen-A BioDesign tutorial fig 20.png2012-10-26T14:27:33Z<p>M.B.ZHOU: </p>
<hr />
<div></div>M.B.ZHOUhttp://2012.igem.org/File:SUSTC-Shenzhen-A_BioDesign_tutorial_fig_19.pngFile:SUSTC-Shenzhen-A BioDesign tutorial fig 19.png2012-10-26T14:25:04Z<p>M.B.ZHOU: </p>
<hr />
<div></div>M.B.ZHOUhttp://2012.igem.org/File:SUSTC-Shenzhen-A_BioDesign_tutorial_fig_18.pngFile:SUSTC-Shenzhen-A BioDesign tutorial fig 18.png2012-10-26T14:24:34Z<p>M.B.ZHOU: </p>
<hr />
<div></div>M.B.ZHOUhttp://2012.igem.org/File:SUSTC-Shenzhen-A_BioDesign_tutorial_fig_17.pngFile:SUSTC-Shenzhen-A BioDesign tutorial fig 17.png2012-10-26T14:20:35Z<p>M.B.ZHOU: </p>
<hr />
<div></div>M.B.ZHOUhttp://2012.igem.org/File:SUSTC-Shenzhen-A_BioDesign_tutorial_fig_16.pngFile:SUSTC-Shenzhen-A BioDesign tutorial fig 16.png2012-10-26T14:19:02Z<p>M.B.ZHOU: </p>
<hr />
<div></div>M.B.ZHOUhttp://2012.igem.org/Team:SUSTC-Shenzhen-A/Biodesign_TutorialTeam:SUSTC-Shenzhen-A/Biodesign Tutorial2012-10-26T14:16:25Z<p>M.B.ZHOU: </p>
<hr />
<div>{{Template:SUSTC_A2}}<br />
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<td><div><br />
<h1 class="title">&nbsp;BioDesign Tutorial</h1>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<ul><br />
<a href="#Folder">Folder page <br />
</a> &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
<br />
<a href="#File">File page <br />
</a>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
<br />
<a href="#Drawing">Drawing page <br />
</a>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
<br />
<a href="#Mail">Mail page <br />
</a> &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
<a href="#Video">Video tutorial<br />
</a> <br />
</ul><br />
<table border="0" cellspacing="0" cellpadding="0"><br />
<tr><br />
<td>&nbsp;</td><br />
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<br />
<br />
<tr><td><a name="Folder" ></a> <br />
<p class="title1">&nbsp;&nbsp;Folder page</p><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/Devidingline_whole.jpg"><br />
<br />
<p><img src="https://static.igem.org/mediawiki/2012/e/e6/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_1.png" valign="top" align="left" width="300" height = "480" style="BORDER:#CCFFCC 5px dashed;margin:10px;" ><img src="https://static.igem.org/mediawiki/2012/b/b7/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_2.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:10px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(1)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Fig.1 is the first page. </p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;In order to create a new project/folder, you should click the ‘add’ button at the top right (Fig.2).</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(2)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
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<p><img src="https://static.igem.org/mediawiki/2012/3/35/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_3.png" valign="top" align="left" height="480" width="300" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(3)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the icon, then click ‘enter’,<br/> it goes into the secondary page (Fig.3).</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
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<br />
</td><br />
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<br />
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<td><a name="File" ></a> <br />
<p class="title1">&nbsp;&nbsp;File page</p><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/Devidingline_whole.jpg"><br />
<p><img src="https://static.igem.org/mediawiki/2012/2/26/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_4.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/6/60/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_5.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:10px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(4)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;The secondary page looks like Fig.4. </p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the button name ‘add’ at the top right to create a new file (Fig.5).</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(5)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
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<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<a name="Drawing"></a><br />
<p class="title1">&nbsp;&nbsp;Drawing page</p><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/Devidingline_whole.jpg"><br />
<p><img src="https://static.igem.org/mediawiki/2012/4/41/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_6.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/5/54/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_7.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(6)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the newly created file, choose ‘edit’ to do the drawing (Fig.6).</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click a object at the bottom to set it on the screen.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(7)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
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<br />
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<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/8/8f/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_8.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/b/b2/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_9.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(8)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the text field next to a part to rename this part.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;For the objects in “comp”, you can zoom them by ywo fingers to change the their sizes.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(9)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
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<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/1/14/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_10.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/f/f6/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_11.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(10)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can rotate the object clockwisely by clicking the rotating button.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can get a vector on the canvas from “part”.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(11)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/f/fd/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_12.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/c/c8/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_13.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(12)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can change the size of the vector by zooming with two fingers.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;If you have add a cell, you may find it covers the vector or other object. Click the “down” button to lower layer.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(13)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/6/63/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_14.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/7/71/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_15.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(14)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can put objects(in”mole. & part.”) on vector by draging it into the vector.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;In “reac.”, you have various choices to connect objects by Bezier curve.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(15)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/6/63/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_14.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/7/71/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_15.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(14)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can put objects(in”mole. & part.”) on vector by draging it into the vector.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;In “reac.”, you have various choices to connect objects by Bezier curve.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(15)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/d/df/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_70.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/9/90/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_71.png" valign="top" align="right" width="300"height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(70)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the button at the top right to save this picture (Fig.70). </p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Come back, you’ll see the icon has change into the saved picture (Fig.71).</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(71)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/e/ef/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_72.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/f/f8/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_73.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(72)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the microscope button in FIle page at the bottom to search a file (Fig.72). </p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbspClick the button at the bottom right to present the files in a table (Fig.73).</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(73)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><a name="Mail" ></a> <br />
<p class="title1">&nbsp;&nbsp;Mail page</p><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/Devidingline_whole.jpg"><br />
<p><img src="https://static.igem.org/mediawiki/2012/b/bf/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_74.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/5/5a/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_75.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:10px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(74)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Come back to the Folder page. Click the envelope button at the bottom right to the file that you want to email to your friend! (Fig.74)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the ‘done’ button. Send the chosen files to your friends (Fig.75)!</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(75)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><a name="More" ></a> <br />
<p class="title1">&nbsp;&nbsp;More</p><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/Devidingline_whole.jpg"><br />
<p><img src="https://static.igem.org/mediawiki/2012/d/dc/BiosearchPage16.png" valign="top" align="left" width="300" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/6/64/BiosearchPage17.png" valign="top" align="right" width="300" style="BORDER:#CCFFCC 5px dashed;margin:10px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(16)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;In the "more" page, you can get some information about synthetic biology and biobricks.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;On the bottom, there is the feedback part.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(17)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/e/ef/BiosearchPage19.png" valign="top" align="left" width="300" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/5/5c/BiosearchPage20.png" valign="top" align="right" width="300" style="BORDER:#CCFFCC 5px dashed;margin:10px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(18)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;The ‘About us’ part show the information of our team.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the ‘Compose the mail’ to send your suggestion to us.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(19)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</br><br />
</br><br />
</br><br />
</br><br />
<a name="Video"></a><br />
<p class="title1">&nbsp;&nbsp;Simplified Video tutorial</p><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/Devidingline_whole.jpg"><br />
<p>&nbsp;&nbsp;</p><br />
<p align="center"><embed src="http://player.youku.com/player.php/sid/XNDY3MDg5NTk2/v.swf" play="true" quality="high" width="540" height="400" align="middle" allowScriptAccess="always" type="application/x-shockwave-flash"></embed></p><a name="video"></a><br />
</td><br />
</tr><br />
<br />
</table><br />
</p><br />
<div></td><br />
</tr><br />
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[[file:footbar.jpg|center]]</div>M.B.ZHOUhttp://2012.igem.org/File:SUSTC-Shenzhen-A_BioDesign_tutorial_fig_15.pngFile:SUSTC-Shenzhen-A BioDesign tutorial fig 15.png2012-10-26T14:14:28Z<p>M.B.ZHOU: </p>
<hr />
<div></div>M.B.ZHOUhttp://2012.igem.org/File:SUSTC-Shenzhen-A_BioDesign_tutorial_fig_14.pngFile:SUSTC-Shenzhen-A BioDesign tutorial fig 14.png2012-10-26T14:12:57Z<p>M.B.ZHOU: </p>
<hr />
<div></div>M.B.ZHOUhttp://2012.igem.org/Team:SUSTC-Shenzhen-A/Biodesign_TutorialTeam:SUSTC-Shenzhen-A/Biodesign Tutorial2012-10-26T14:11:41Z<p>M.B.ZHOU: </p>
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border-width: thin;<br />
border-top-color:#DCDCDC;<br />
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font-size: 14px;<br />
color:#333;<br />
font-family: Tahoma, Geneva;<br />
}<br />
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<body ><br />
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<table width="899" border="0" cellspacing="0px" cellpadding="0px" align="center"><br />
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<div> <tr><br />
<td><div><br />
<h1 class="title">&nbsp;BioDesign Tutorial</h1>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<ul><br />
<a href="#Folder">Folder page <br />
</a> &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
<br />
<a href="#File">File page <br />
</a>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
<br />
<a href="#Drawing">Drawing page <br />
</a>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
<br />
<a href="#Mail">Mail page <br />
</a> &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
<a href="#Video">Video tutorial<br />
</a> <br />
</ul><br />
<table border="0" cellspacing="0" cellpadding="0"><br />
<tr><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<br />
<tr><td><a name="Folder" ></a> <br />
<p class="title1">&nbsp;&nbsp;Folder page</p><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/Devidingline_whole.jpg"><br />
<br />
<p><img src="https://static.igem.org/mediawiki/2012/e/e6/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_1.png" valign="top" align="left" width="300" height = "480" style="BORDER:#CCFFCC 5px dashed;margin:10px;" ><img src="https://static.igem.org/mediawiki/2012/b/b7/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_2.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:10px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(1)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Fig.1 is the first page. </p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;In order to create a new project/folder, you should click the ‘add’ button at the top right (Fig.2).</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(2)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
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<p><img src="https://static.igem.org/mediawiki/2012/3/35/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_3.png" valign="top" align="left" height="480" width="300" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(3)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the icon, then click ‘enter’,<br/> it goes into the secondary page (Fig.3).</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
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<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><a name="File" ></a> <br />
<p class="title1">&nbsp;&nbsp;File page</p><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/Devidingline_whole.jpg"><br />
<p><img src="https://static.igem.org/mediawiki/2012/2/26/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_4.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/6/60/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_5.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:10px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(4)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;The secondary page looks like Fig.4. </p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the button name ‘add’ at the top right to create a new file (Fig.5).</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(5)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
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<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
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<br />
<tr><br />
<td><br />
<a name="Drawing"></a><br />
<p class="title1">&nbsp;&nbsp;Drawing page</p><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/Devidingline_whole.jpg"><br />
<p><img src="https://static.igem.org/mediawiki/2012/4/41/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_6.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/5/54/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_7.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(6)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the newly created file, choose ‘edit’ to do the drawing (Fig.6).</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click a object at the bottom to set it on the screen.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(7)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
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</td><br />
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<br />
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<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/8/8f/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_8.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/b/b2/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_9.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(8)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the text field next to a part to rename this part.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;For the objects in “comp”, you can zoom them by ywo fingers to change the their sizes.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(9)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/1/14/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_10.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/f/f6/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_11.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(10)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can rotate the object clockwisely by clicking the rotating button.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can get a vector on the canvas from “part”.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(11)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
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</td><br />
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<br />
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<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/f/fd/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_12.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/c/c8/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_13.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(12)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;You can change the size of the vector by zooming with two fingers.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;If you have add a cell, you may find it covers the vector or other object. Click the “down” button to lower layer.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(13)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
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<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/d/df/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_70.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/9/90/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_71.png" valign="top" align="right" width="300"height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(70)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the button at the top right to save this picture (Fig.70). </p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Come back, you’ll see the icon has change into the saved picture (Fig.71).</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(71)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
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<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
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<br />
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<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/e/ef/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_72.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/f/f8/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_73.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(72)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the microscope button in FIle page at the bottom to search a file (Fig.72). </p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbspClick the button at the bottom right to present the files in a table (Fig.73).</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(73)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
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<p>&nbsp;&nbsp;</p><br />
<br/><br />
<br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</td><br />
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<td><a name="Mail" ></a> <br />
<p class="title1">&nbsp;&nbsp;Mail page</p><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/Devidingline_whole.jpg"><br />
<p><img src="https://static.igem.org/mediawiki/2012/b/bf/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_74.png" valign="top" align="left" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/5/5a/SUSTC-Shenzhen-A_BioDesign_tutorial_fig_75.png" valign="top" align="right" width="300" height="480" style="BORDER:#CCFFCC 5px dashed;margin:10px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(74)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Come back to the Folder page. Click the envelope button at the bottom right to the file that you want to email to your friend! (Fig.74)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the ‘done’ button. Send the chosen files to your friends (Fig.75)!</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(75)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
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<p>&nbsp;&nbsp;</p><br />
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<br />
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<td><a name="More" ></a> <br />
<p class="title1">&nbsp;&nbsp;More</p><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/Devidingline_whole.jpg"><br />
<p><img src="https://static.igem.org/mediawiki/2012/d/dc/BiosearchPage16.png" valign="top" align="left" width="300" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/6/64/BiosearchPage17.png" valign="top" align="right" width="300" style="BORDER:#CCFFCC 5px dashed;margin:10px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(16)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;In the "more" page, you can get some information about synthetic biology and biobricks.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;On the bottom, there is the feedback part.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(17)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
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<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
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</td><br />
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<td><br />
<p><img src="https://static.igem.org/mediawiki/2012/e/ef/BiosearchPage19.png" valign="top" align="left" width="300" style="BORDER:#CCFFCC 5px dashed;margin:5px;" ><img src="https://static.igem.org/mediawiki/2012/5/5c/BiosearchPage20.png" valign="top" align="right" width="300" style="BORDER:#CCFFCC 5px dashed;margin:10px;" ></p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;<--(18)</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;The ‘About us’ part show the information of our team.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;Click the ‘Compose the mail’ to send your suggestion to us.</p><br />
<p>&nbsp;&nbsp;</p><br />
<p class="title">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(19)--></p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
<p>&nbsp;&nbsp;</p><br />
</br><br />
</br><br />
</br><br />
</br><br />
<a name="Video"></a><br />
<p class="title1">&nbsp;&nbsp;Simplified Video tutorial</p><br />
<img src="https://static.igem.org/mediawiki/2012/0/0b/Devidingline_whole.jpg"><br />
<p>&nbsp;&nbsp;</p><br />
<p align="center"><embed src="http://player.youku.com/player.php/sid/XNDY3MDg5NTk2/v.swf" play="true" quality="high" width="540" height="400" align="middle" allowScriptAccess="always" type="application/x-shockwave-flash"></embed></p><a name="video"></a><br />
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[[file:footbar.jpg|center]]</div>M.B.ZHOUhttp://2012.igem.org/File:SUSTC-Shenzhen-A_BioDesign_tutorial_fig_13.pngFile:SUSTC-Shenzhen-A BioDesign tutorial fig 13.png2012-10-26T14:08:28Z<p>M.B.ZHOU: </p>
<hr />
<div></div>M.B.ZHOU