Team:KAIST Korea/Notebook Labnote/2012 10

From 2012.igem.org

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<section id="1">
<section id="1">
<div class="date">October 1<sup>st</sup> 2012</div></br>
<div class="date">October 1<sup>st</sup> 2012</div></br>
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<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:260px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:35px;float:bottom;"id="packman" src='https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png'/>　PACKMAN<img style="height:3px;width:260px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
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<div class="note-title">Plasmid gel electrophoresis</div>
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<div id="content_note" >
<div id="content_note" >
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<span id="little">No Special Event!</span>
 
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</br>
 
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<b>Results</b></br></br>
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<div align="center"><img id="figure" alt="1_1001Fig1" src="https://static.igem.org/mediawiki/2012/4/49/KAIST_PACKMAN_1001_Fig1.png"></img></div>
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<div style="clear:both;"></div>
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</br>
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<span id="little">Plasmid of Gibson fragment 1 (sample 1~10) were run on the gel to see if there is any problem with plasmid mini-prep process. </span>
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</br></br>
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<b>Discussions</b></br></br>
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<span id="little">It seems that this vector is supercoiled form, of which the size is about 10000bp.</span>
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</br>
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</div>
</div>
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<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
</section>
</section>
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<section id="2">
<section id="2">
<div class="date">October 2<sup>nd</sup> 2012</div></br>
<div class="date">October 2<sup>nd</sup> 2012</div></br>
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<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:269px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/>　Flip Flop<img style="height:3px;width:269px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
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<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:260px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/>　Flip Flop<img style="height:3px;width:260px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
<div class="note-title">bFMO template preparation with new primer</div>
<div class="note-title">bFMO template preparation with new primer</div>
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<span id="little"></br>We have found that our bFMO sequence was wrong. So that we have ordered new primers with right sequence.</br></span>
<span id="little"></br>We have found that our bFMO sequence was wrong. So that we have ordered new primers with right sequence.</br></span>
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</br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br>
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<div class="note-title"> OE PCR template preparation</div>
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<div id="content_note" >
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<b>Results</b></br></br>
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<div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/7/7f/121002_OE_pcr.PNG"></img></div>
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<div style="clear:both;"></div>
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</div>
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</br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br>
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<div class="note-title"> pAutoIntegrase cloning check</div>
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<div id="content_note" >
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<b>Results</b></br></br>
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<div align="center"><img id="figure" alt="0818Fig1" src="https://static.igem.org/mediawiki/2012/7/72/121002_pAutoIntegrase_cloning_check.PNG"></img></div>
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<div style="clear:both;"></div>
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</br></br>
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<b>Discussion</b></br></br>
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<span id="little">We have checked the result of cloning with various ways but we can’t sure about the success of cloning.</span>
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</div>
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</br><div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:260px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:35px;float:bottom;"id="packman" src='https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png'/>　PACKMAN<img style="height:3px;width:260px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
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<div class="note-title">Restriction enzyme cut</div>
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<div id="content_note" >
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<b>Results</b></br></br>
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<span id="little">Fragment 1 was digested with Hind III and Not I. Digestion of vector generates four fragments, the size of each fragments are as follows: 15280bp, 6593bp, 5748bp, 3000bp. Fragment 2 was digested with Not I and Xho I. Digestion of vector generates three fragments. The size of each fragments are as follows : 11213bp, 9010bp, 2207bp. </span>
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</br>
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<span id ="little">Table below is composition of each enzyme reaction.</span></br></br>
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<div align="center"><img id="figure" alt="1_1002Fig1" src="https://static.igem.org/mediawiki/2012/1/17/KAIST_PACKMAN_1002_Fig1.png"></img></div>
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<div style="clear:both;"></div>
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</br>
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<div align="center"><img id="figure" alt="1_1002Fig2" src="https://static.igem.org/mediawiki/2012/a/a5/KAIST_PACKMAN_1002_Fig2.png"></img></div>
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<div style="clear:both;"></div>
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</br>
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<b>Discussions</b></br></br>
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<span id="little">Different from our prediction, bands appeared at the wrong size, about the exact size of the vector. Therefore, we could conclude that colonies F1 1~10 and F2 1~5 contain background vectors. </span>
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</br>
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</div>
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</br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br>
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<div class="note-title">Restriction enzyme cut _ gel 2</div>
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<div id="content_note" >
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<b>Results</b></br></br>
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<div align="center"><img id="figure" alt="1_1002Fig3" src="https://static.igem.org/mediawiki/2012/2/24/KAIST_PACKMAN_1002_Fig3.png"></img></div>
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<div style="clear:both;"></div>
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</br>
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<b>Discussions</b></br></br>
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<span id="little">For colony 6, 8, and 10, there appeared correct size of whole vector size. It seemed that Not I or Xho I was not active on the reaction condition and only one of those two enzymes cut circular DNA into linear DNA. </span>
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</br>
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</div>
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</br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br>
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<div class="note-title">Pre-culture of Gibson F1 colonies</div>
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<div id="content_note" >
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<span id="little">Additional 10 colonies for fragment 1 were picked, and culture into 3mL LB media containing 30ul Kanamycin.</span>
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</br>
</div>
</div>
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
</section>
</section>
 +
<section id="3">
<section id="3">
<div class="date">October 3<sup>rd</sup> 2012</div></br>
<div class="date">October 3<sup>rd</sup> 2012</div></br>
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<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:269px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/>　Flip Flop<img style="height:3px;width:269px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
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<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:260px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/>　Flip Flop<img style="height:3px;width:260px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
<div class="note-title">Visualization of inversion from GFP to RFP</div>
<div class="note-title">Visualization of inversion from GFP to RFP</div>
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<div id="content_note" >
<div id="content_note" >
-
<b>Procedure</b></br></br>
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<b>Procedure</b></br>
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<span id="little">We sampled the cell every 3hr. We induced one group of samples and un-induced another group of samples as a control. We took the picture of cell with confocal microscope at absorbance 500nm for GFP expression and 600nm for RFP expression.  </br>
+
<span id="little">We sampled the cell every 3hr. We induced one group of samples and un-induced another group of samples as a control. We took the picture of cell with confocal microscope at absorbance 500nm for GFP expression and 600nm for RFP expression.  </br></br>
</span>
</span>
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<div style="clear:both;"></div>
<div style="clear:both;"></div>
</div>
</div>
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<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:260px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:35px;float:bottom;"id="packman" src='https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png'/>　PACKMAN<img style="height:3px;width:260px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
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<div class="note-title">Vector preparation and Enzyme cut</div>
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<div id="content_note" >
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<span id="little">For each cultured cells, vectors were mini-prepped, and we did enzyme cut with those vectors. </span>
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</br>
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<span id ='little'>Table below is composition of each enzyme reaction.</span></br></br>
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<b>Results</b></br></br>
 +
 
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<div align="center"><img id="figure" alt="1_1003Fig1" src="https://static.igem.org/mediawiki/2012/e/ee/KAIST_PACKMAN_1003_Fig1.png"></img></div>
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<div style="clear:both;"></div>
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</br>
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<span id="little"></span>
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</br>
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</div>
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
</section>
</section>
<section id="4">
<section id="4">
<div class="date">October 4<sup>th</sup> 2012</div></br>
<div class="date">October 4<sup>th</sup> 2012</div></br>
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<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:260px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:35px;float:bottom;"id="packman" src='https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png'/>　PACKMAN<img style="height:3px;width:260px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
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<div class="note-title">Enzyme cut result</div>
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<div id="content_note" >
<div id="content_note" >
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<span id="little">No Special Event!</span>
 
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</br>
 
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<b>Results</b></br></br>
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<div align="center"><img id="figure" alt="1_1004Fig1" src="https://static.igem.org/mediawiki/2012/5/5d/KAIST_PACKMAN_1004_Fig1.png"></img></div>
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<div style="clear:both;"></div>
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</br>
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<span id="little">It seems there are only background vectors in the cells. </span>
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</br>
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</div>
</div>
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<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
</section>
</section>
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<div class="date">October 5<sup>th</sup> 2012</div></br>
<div class="date">October 5<sup>th</sup> 2012</div></br>
<div id="content_note" >
<div id="content_note" >
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<span id="little">Regional Jamboree at HKUST!!!!</span>
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<span id="little">No Special Event!</span>
</br>
</br>
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<div class="date">October 6<sup>th</sup> 2012</div></br>
<div class="date">October 6<sup>th</sup> 2012</div></br>
<div id="content_note" >
<div id="content_note" >
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<span id="little">Regional Jamboree at HKUST!!!!</span>
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<span id="little">No Special Event!</span>
</br>
</br>
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<div class="date">October 7<sup>th</sup> 2012</div></br>
<div class="date">October 7<sup>th</sup> 2012</div></br>
<div id="content_note" >
<div id="content_note" >
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<span id="little">Regional Jamboree at HKUST!!!!</span>
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<span id="little">No Special Event!</span>
</br>
</br>
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<section id="8">
<section id="8">
<div class="date">October 8<sup>th</sup> 2012</div></br>
<div class="date">October 8<sup>th</sup> 2012</div></br>
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<div id="content_note" >
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<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:260px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:35px;float:bottom;"id="packman" src='https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png'/>　PACKMAN<img style="height:3px;width:260px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
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<span id="little">No Special Event!</span>
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</br>
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<div class="note-title">Pre-culture of Gibson assembly fragment1 containing colony</div>
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<div id="content_note" >
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<b>Results</b></br></br>
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<span id="little">Colonies number 16 through 25 were cultured in 3mL LB containing 1000x Kanamycin (30ul). Cells were cultured about 16 hours and vector was mini-prepped.</span>
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</br>
</div>
</div>
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<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
</section>
</section>
 +
<section id="9">
<section id="9">
<div class="date">October 9<sup>th</sup> 2012</div></br>
<div class="date">October 9<sup>th</sup> 2012</div></br>
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<div id="content_note" >
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<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:260px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:35px;float:bottom;"id="packman" src='https://static.igem.org/mediawiki/2012/0/03/KAIST_PACKMAN.png'/>　PACKMAN<img style="height:3px;width:260px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
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<span id="little">No Special Event!</span>
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</br>
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<div class="note-title">Vector mini-prep and enzyme cut of prepped vector.</div>
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<div id="content_note" >
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<b>Results</b></br></br>
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<span id="little">Each vector was prepped and cut with HindIII, NotI to see if proper assembly product has formed. </span></br>
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<span id='little'>Table below is composition of each enzyme reaction.</span></br>
 +
 +
 +
</br>
 +
 +
<div align="center"><img id="figure" alt="1_1009Fig1" src="https://static.igem.org/mediawiki/2012/c/c9/KAIST_PACKMAN_1009_Fig1.png"></img></div>
 +
<div style="clear:both;"></div>
 +
 +
</br>
 +
 +
</div>
</div>
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
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<section id="10">
<section id="10">
<div class="date">October 10<sup>th</sup> 2012</div></br>
<div class="date">October 10<sup>th</sup> 2012</div></br>
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<div id="content_note" >
+
</br><div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:260px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/>　Flip Flop<img style="height:3px;width:260px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
-
<span id="little">No Special Event!</span>
+
 +
<div class="note-title">LuxI and Xis OE template preparation</div>
 +
 
 +
<div id="content_note" >
 +
<b>Results</b></br></br>
 +
 
 +
<div align="center"><img id="figure" alt="2_1010Fig1" src="https://static.igem.org/mediawiki/2012/3/39/KAIST_LABNOTE101012_Fig1.png"></img></div>
 +
<div style="clear:both;"></div>
 +
 
</br>
</br>
 +
</div>
 +
</br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br>
 +
 +
<div class="note-title">pAutoIntegrase cloning check and Xis template vector prep</div>
 +
 +
<div id="content_note" >
 +
 +
<b>Results</b></br></br>
 +
 +
<div align="center"><img id="figure" alt="2_1010Fig2" src="https://static.igem.org/mediawiki/2012/4/4f/KAIST_LABNOTE101012_Fig2.png"></img></div>
 +
<div style="clear:both;"></div>
 +
 +
</br>
 +
 +
<span id="little">pAutoIntegrase 1-10 – HindIII cut</br></span>
 +
<span id="little">pGEM-Xis 1-2 – EcoRI cut</br></span>
 +
 +
 +
</br>
</div>
</div>
 +
 +
</br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br>
 +
 +
<div class="note-title">insert DNA preparation with OE PCR</div>
 +
 +
<div id="content_note" >
 +
 +
<b>Results</b></br></br>
 +
 +
<div align="center"><img id="figure" alt="2_1010Fig3" src="https://static.igem.org/mediawiki/2012/d/df/KAIST_LABNOTE101012_Fig3.png"></img></div>
 +
<div style="clear:both;"></div>
 +
 +
</br>
 +
 +
<span id="little">┗ pAutoSimple(1-3) and pAutoSimpleInverted(4-6)</span></br>
 +
 +
</br>
 +
<div align="center"><img id="figure" alt="2_1010Fig4" src="https://static.igem.org/mediawiki/2012/7/7d/KAIST_LABNOTE101012_Fig4.png"></img></div>
 +
<div style="clear:both;"></div>
 +
 +
</br>
 +
 +
<span id="little">┗ pAutoSimple and pAutoSimpleInverted (1 and 2, respectively)/ pAuto(1-3) and pAutoInverted(4-6)</span>
 +
 +
</br>
 +
</div>
 +
 +
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
Line 336: Line 552:
<section id="11">
<section id="11">
<div class="date">October 11<sup>th</sup> 2012</div></br>
<div class="date">October 11<sup>th</sup> 2012</div></br>
-
<div id="content_note" >
+
</br><div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:260px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/>　Flip Flop<img style="height:3px;width:260px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
-
<span id="little">No Special Event!</span>
+
-
</br>
+
 +
<div class="note-title">Additional templates</div>
 +
 +
<div id="content_note" >
 +
 +
<b>Results</b></br></br>
 +
 +
<div align="center"><img id="figure" alt="2_1011Fig1" src="https://static.igem.org/mediawiki/2012/2/2d/KAIST_LABNOTE101112_Fig1.png"></img></div>
 +
<div style="clear:both;"></div>
 +
 +
</br>
 +
<div align='center'>
 +
<span id="little">┗ Integrase and Excisionase template</span></div>
 +
 +
</br>
 +
 +
</div>
</div>
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
Line 365: Line 595:
<section id="14">
<section id="14">
<div class="date">October 14<sup>th</sup> 2012</div></br>
<div class="date">October 14<sup>th</sup> 2012</div></br>
-
<div id="content_note" >
+
</br><div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:260px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/>　Flip Flop<img style="height:3px;width:260px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
-
<span id="little">No Special Event!</span>
+
-
</br>
+
 +
<div class="note-title">pAutoIntegrase Cloning</div>
 +
 +
<div id="content_note" >
 +
 +
<span id="little">Insert DNA – OE PCR</span>
 +
</br></br>
 +
<b>Results</b></br></br>
 +
 +
<div align="center"><img id="figure" alt="2_1014Fig1" src="https://static.igem.org/mediawiki/2012/1/12/KAIST_LABNOTE101412_Fig1.png"></img></div>
 +
<div style="clear:both;"></div>
 +
 +
</br>
 +
<div align='center'>
 +
<span id="little">Cloning into pSB4A5 is performed with gel extracted DNA fragment.</span></div>
 +
 +
</br>
 +
 +
</div>
</div>
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
Line 384: Line 630:
<section id="16">
<section id="16">
<div class="date">October 16<sup>th</sup> 2012</div></br>
<div class="date">October 16<sup>th</sup> 2012</div></br>
 +
</br><div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:260px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/>　Flip Flop<img style="height:3px;width:260px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
 +
 +
<div class="note-title">pAutoSimple inserts nested PCR and aiiA template</div>
 +
<div id="content_note" >
<div id="content_note" >
-
<span id="little">No Special Event!</span>
 
-
</br>
 
 +
<b>Results</b></br></br>
 +
 +
<div align="center"><img id="figure" alt="2_1016Fig1" src="https://static.igem.org/mediawiki/2012/c/cb/KAIST_LABNOTE101612_Fig1.png"></img></div>
 +
<div style="clear:both;"></div>
 +
 +
</br>
</div>
</div>
 +
</br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br>
 +
 +
<div class="note-title">pAuto, pAutoInverted, pAutoSimple, pAutoSimpleInverted miniprep and RE cut</div>
 +
 +
<div id="content_note" >
 +
 +
<b>Results</b></br></br>
 +
 +
<div align="center"><img id="figure" alt="2_1016Fig2" src="https://static.igem.org/mediawiki/2012/c/c4/KAIST_LABNOTE101612_Fig2.png"></img></div>
 +
<div style="clear:both;"></div>
 +
 +
</br>
 +
 +
<span id="little">single digestion with HindIII.</span>
 +
 +
</br>
 +
</div>
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
Line 395: Line 666:
<section id="17">
<section id="17">
<div class="date">October 17<sup>th</sup> 2012</div></br>
<div class="date">October 17<sup>th</sup> 2012</div></br>
 +
</br><div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:260px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/>　Flip Flop<img style="height:3px;width:260px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
 +
 +
<div class="note-title">pAuto OE PCR and Xis, simple bFMO, and bFMO template preparation</div>
<div id="content_note" >
<div id="content_note" >
-
<span id="little">No Special Event!</span>
+
 +
<b>Results</b></br></br>
 +
 
 +
<div align="center"><img id="figure" alt="2_1017Fig1" src="https://static.igem.org/mediawiki/2012/9/9e/KAIST_LABNOTE101712_Fig1.png"></img></div>
 +
<div style="clear:both;"></div>
 +
 
</br>
</br>
 +
 +
<span id="little">sizes ok</span>
 +
 +
</br>
 +
 +
 +
</div>
 +
</br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br>
 +
 +
<div class="note-title">pAuto insert DNA preparation</div>
 +
 +
<div id="content_note" >
 +
 +
<b>Results</b></br></br>
 +
 +
<div align="center"><img id="figure" alt="2_1017Fig2" src="https://static.igem.org/mediawiki/2012/5/56/KAIST_LABNOTE101712_Fig2.png"></img></div>
 +
<div style="clear:both;"></div>
 +
 +
</br>
 +
<div align='center'>
 +
<span id="little">intermediate fragment nested PCR</span></div>
 +
 +
</br>
 +
<div align="center"><img id="figure" alt="2_1017Fig3" src="https://static.igem.org/mediawiki/2012/7/79/KAIST_LABNOTE101712_Fig3.png"></img></div>
 +
<div style="clear:both;"></div>
 +
 +
</br>
 +
<div align='center'>
 +
<span id="little">Full pAuto insert DNA construct OE PCR</span></div>
 +
 +
</br>
 +
</div>
</div>
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
Line 405: Line 716:
<section id="18">
<section id="18">
<div class="date">October 18<sup>th</sup> 2012</div></br>
<div class="date">October 18<sup>th</sup> 2012</div></br>
-
<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:269px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/>　Flip Flop<img style="height:3px;width:269px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
+
</br><div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:260px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/>　Flip Flop<img style="height:3px;width:260px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
 +
 +
<div class="note-title">pAutoIntegrase enz cut check with NotI</div>
 +
 
 +
<div id="content_note" >
 +
 +
<b>Results</b></br></br>
 +
 
 +
<div align="center"><img id="figure" alt="2_1018Fig1" src="https://static.igem.org/mediawiki/2012/6/66/KAIST_LABNOTE101812_Fig1.png"></img></div>
 +
<div style="clear:both;"></div>
 +
 
 +
</br>
 +
 
 +
<span id="little">Lane 3, 6, 8 seem to be cloned.</span>
 +
 +
</br>
 +
 
 +
 
 +
</div>
 +
</br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br>
<div class="note-title">Cell culture in 96-well plate for Indigo expression time optimization</div>
<div class="note-title">Cell culture in 96-well plate for Indigo expression time optimization</div>
Line 425: Line 755:
-
<span id="little"></br>We subtracted absorbance value of pure LB from value of samples and we set all samples to have same amount of cell using O.D value </br></br></span>
+
<span id="little"></br></br>We subtracted absorbance value of pure LB from value of samples and we set all samples to have same amount of cell using O.D value </br>
 +
Graph shown below is indigo intensity per cell. After 3 days incubation, indigo seems to be degraded and all the samples seem to have almost same indigo level.</br></br></span>
 +
 
 +
<div align="center"><img id="figure" alt="0818Fig1" style="width:670px;"src="https://static.igem.org/mediawiki/2012/f/fc/Tecan_indigo-1.PNG"></img></div>
 +
<div style="clear:both;"></div></br>
 +
 
 +
<span id="little"></br>We excluded 0hr data from the graph to see teh rising tendency clearly.</br></br></span>
 +
 
 +
<div align="center"><img id="figure" style="width:670px;"src="https://static.igem.org/mediawiki/2012/0/06/Tecan_indigo-2.PNG"></img></div>
 +
<div style="clear:both;"></div>
-
<b>Discussion</b></br></br>
+
<b></br>Discussion</b></br>
<span id="little"></br>This data shows that the cell successfully produces indigo. We can use this gene as a reporter of our module. However, we only checked the increase of indigo. It may decrease after prolonged culture. We should do additional experiments to get the peak in the graph and optimize the production time of indigo. </br></span>
<span id="little"></br>This data shows that the cell successfully produces indigo. We can use this gene as a reporter of our module. However, we only checked the increase of indigo. It may decrease after prolonged culture. We should do additional experiments to get the peak in the graph and optimize the production time of indigo. </br></span>
Line 433: Line 772:
</div>
</div>
 +
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
Line 438: Line 778:
<section id="19">
<section id="19">
<div class="date">October 19<sup>th</sup> 2012</div></br>
<div class="date">October 19<sup>th</sup> 2012</div></br>
 +
</br><div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:260px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/>　Flip Flop<img style="height:3px;width:260px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
 +
 +
<div class="note-title">pAutoIntegrase Alternative sequencing with fragments amplification</div>
 +
<div id="content_note" >
<div id="content_note" >
-
<span id="little">No Special Event!</span>
 
-
</br>
 
 +
<b>Results</b></br></br>
 +
 +
<div align="center"><img id="figure" alt="2_1019Fig1" src="https://static.igem.org/mediawiki/2012/9/9d/KAIST_LABNOTE101912_Fig1.png"></img></div>
 +
<div style="clear:both;"></div>
 +
 +
</br>
 +
 +
</div>
</div>
-
+
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
-
+
-
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
+
</section>
</section>
Line 487: Line 835:
<section id="24">
<section id="24">
<div class="date">October 24<sup>th</sup> 2012</div></br>
<div class="date">October 24<sup>th</sup> 2012</div></br>
-
<div id="content_note" >
+
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<span id="little">No Special Event!</span>
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</br>
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<div class="note-title">OE template preparation</div>
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<div id="content_note" >
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<b>Results</b></br></br>
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<div align="center"><img id="figure" alt="2_1024Fig1" src="https://static.igem.org/mediawiki/2012/9/96/KAIST_LABNOTE102412_Fig1.png"></img></div>
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<div style="clear:both;"></div>
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<div align = 'center'>
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<span id="little">gp47, aiiA, LuxI, bFMO for simple and pAuto</span></div>
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</br>
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<div align="center"><img id="figure" alt="2_1024Fig1" src="https://static.igem.org/mediawiki/2012/5/52/KAIST_LABNOTE102412_Fig2.png"></img></div>
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<div style="clear:both;"></div>
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<div align = 'center'>
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<span id="little">promoter and Inverted one</span></div>
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</br>
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</div>
</div>
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
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<div class="date">October 25<sup>th</sup> 2012</div></br>
<div class="date">October 25<sup>th</sup> 2012</div></br>
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<div id="linegray" align="center" style="font-size:15px; font-weight:bold; color:#888;"><img style="height:3px;width:260px;margin:0px 5px 0px 0px; "src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/><img style="width:40px; height:28px;float:bottom;"id="flipflop" src='https://static.igem.org/mediawiki/2012/2/22/KAIST_FlipFlop.png'/>　Flip Flop<img style="height:3px;width:260px; margin:0px 0px 0px 5px;"src="https://static.igem.org/mediawiki/2012/1/1b/KAIST_horizontal_gray.png"/></div></br>
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<span id="little">No Special Event!</span>
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<div class="note-title">pAuto intermediate fragment OE PCR</div>
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<b>Results</b></br></br>
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<div align="center"><img id="figure" alt="1025Fig1" src="https://static.igem.org/mediawiki/2012/f/f1/KAIST_LABNOTE102512_Fig1.png"></img></div>
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</br><div align="center"><img style="width:670px; height:2px;" src="https://static.igem.org/mediawiki/2012/7/7f/Horizontal_line_kaist.png"></img></div></br>
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<div class="note-title">OE PCR with new primer</div>
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<div id="content_note" >
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<b>Results</b></br></br>
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<div align="center"><img id="figure" alt="2_1025Fig2" src="https://static.igem.org/mediawiki/2012/a/a9/KAIST_LABNOTE102512_Fig2.png"></img></div>
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<div style="clear:both;"></div>
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</br>
</br>
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<div align='center'>
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<span id="little">pAutoSimple/inverted</span></div>
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</br>
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<div align="center"><img id="figure" alt="2_1025Fig2" src="https://static.igem.org/mediawiki/2012/9/9d/KAIST_LABNOTE102512_Fig3.png"></img></div>
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<div style="clear:both;"></div>
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<div align='center'>
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<span id="little">pAuto/inverted</span></div>
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</br>
</div>
</div>
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
<a id="back_to_the_calender" style="font-size:12px;color:#111;text-decoration:none;float:right;margin:20px 0px 0px 5px;" href="https://2012.igem.org/wiki/index.php?title=Team:KAIST_Korea/Notebook_Labnote"><img src="https://static.igem.org/mediawiki/2012/e/e3/KAIST_calendar_icon.png" style="width:60px;height:60px;display:none;"/>Back to the Calendar</a>
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Latest revision as of 03:01, 27 October 2012

KAIST Korea 2012 iGEM

Notebook : Labnote-October

Labnote

October

October 1st 2012

　PACKMAN

Plasmid gel electrophoresis
Results

1_1001Fig1

Plasmid of Gibson fragment 1 (sample 1~10) were run on the gel to see if there is any problem with plasmid mini-prep process.

Discussions

It seems that this vector is supercoiled form, of which the size is about 10000bp.
Back to the Calendar
October 2nd 2012

　Flip Flop

bFMO template preparation with new primer
Results

0818Fig1

We have found that our bFMO sequence was wrong. So that we have ordered new primers with right sequence.

OE PCR template preparation
Results

0818Fig1


pAutoIntegrase cloning check
Results

0818Fig1


Discussion

We have checked the result of cloning with various ways but we can’t sure about the success of cloning.

　PACKMAN

Restriction enzyme cut
Results

Fragment 1 was digested with Hind III and Not I. Digestion of vector generates four fragments, the size of each fragments are as follows: 15280bp, 6593bp, 5748bp, 3000bp. Fragment 2 was digested with Not I and Xho I. Digestion of vector generates three fragments. The size of each fragments are as follows : 11213bp, 9010bp, 2207bp.
Table below is composition of each enzyme reaction.

1_1002Fig1

1_1002Fig2

Discussions

Different from our prediction, bands appeared at the wrong size, about the exact size of the vector. Therefore, we could conclude that colonies F1 1~10 and F2 1~5 contain background vectors.


Restriction enzyme cut _ gel 2
Results

1_1002Fig3

Discussions

For colony 6, 8, and 10, there appeared correct size of whole vector size. It seemed that Not I or Xho I was not active on the reaction condition and only one of those two enzymes cut circular DNA into linear DNA.


Pre-culture of Gibson F1 colonies
Additional 10 colonies for fragment 1 were picked, and culture into 3mL LB media containing 30ul Kanamycin.
Back to the Calendar
October 3rd 2012

　Flip Flop

Visualization of inversion from GFP to RFP
Procedure


bfmo gene is constructed in the vector(pJ401, Km registance) and transformed into DH5α.
50λ cell from stock is inoculated in 5mL LB


Visualization of inversion from GFP to RFP
Procedure
We sampled the cell every 3hr. We induced one group of samples and un-induced another group of samples as a control. We took the picture of cell with confocal microscope at absorbance 500nm for GFP expression and 600nm for RFP expression.

Results

0818Fig1


Discussion

We obtained proper data. Un-induced sample doesn’t show measurable RFP expression throughout the culture. However, induced sample shows the increase of RFP expression. Increase of GFP level in induced sample is due to the stability of GFP and time delay before inversion.


Insert DNA preparation with OE pcr
Results

0818Fig1


Cloning
Results

0818Fig1
0818Fig1
　PACKMAN

Vector preparation and Enzyme cut
For each cultured cells, vectors were mini-prepped, and we did enzyme cut with those vectors.
Table below is composition of each enzyme reaction.

Results

1_1003Fig1


Back to the Calendar
October 4th 2012

　PACKMAN

Enzyme cut result
Results

1_1004Fig1

It seems there are only background vectors in the cells.
Back to the Calendar
October 5th 2012

No Special Event!
Back to the Calendar
October 6th 2012

No Special Event!
Back to the Calendar
October 7th 2012

No Special Event!
Back to the Calendar
October 8th 2012

　PACKMAN

Pre-culture of Gibson assembly fragment1 containing colony
Results

Colonies number 16 through 25 were cultured in 3mL LB containing 1000x Kanamycin (30ul). Cells were cultured about 16 hours and vector was mini-prepped.
Back to the Calendar
October 9th 2012

　PACKMAN

Vector mini-prep and enzyme cut of prepped vector.
Results

Each vector was prepped and cut with HindIII, NotI to see if proper assembly product has formed.
Table below is composition of each enzyme reaction.

1_1009Fig1

Back to the Calendar
October 10th 2012


　Flip Flop

LuxI and Xis OE template preparation
Results

2_1010Fig1



pAutoIntegrase cloning check and Xis template vector prep
Results

2_1010Fig2

pAutoIntegrase 1-10 – HindIII cut
 pGEM-Xis 1-2 – EcoRI cut


insert DNA preparation with OE PCR
Results

2_1010Fig3

┗ pAutoSimple(1-3) and pAutoSimpleInverted(4-6)

2_1010Fig4

┗ pAutoSimple and pAutoSimpleInverted (1 and 2, respectively)/ pAuto(1-3) and pAutoInverted(4-6)
Back to the Calendar
October 11th 2012


　Flip Flop

Additional templates
Results

2_1011Fig1

┗ Integrase and Excisionase template

Back to the Calendar
October 12th 2012

No Special Event!
Back to the Calendar
October 13th 2012

No Special Event!
Back to the Calendar
October 14th 2012


　Flip Flop

pAutoIntegrase Cloning
Insert DNA – OE PCR

Results

2_1014Fig1

Cloning into pSB4A5 is performed with gel extracted DNA fragment.

Back to the Calendar
October 15th 2012

No Special Event!
Back to the Calendar
October 16th 2012


　Flip Flop

pAutoSimple inserts nested PCR and aiiA template
Results

2_1016Fig1



pAuto, pAutoInverted, pAutoSimple, pAutoSimpleInverted miniprep and RE cut
Results

2_1016Fig2

single digestion with HindIII.
Back to the Calendar
October 17th 2012


　Flip Flop

pAuto OE PCR and Xis, simple bFMO, and bFMO template preparation
Results

2_1017Fig1

sizes ok


pAuto insert DNA preparation
Results

2_1017Fig2

intermediate fragment nested PCR

2_1017Fig3

Full pAuto insert DNA construct OE PCR

Back to the Calendar
October 18th 2012


　Flip Flop

pAutoIntegrase enz cut check with NotI
Results

2_1018Fig1

Lane 3, 6, 8 seem to be cloned.


Cell culture in 96-well plate for Indigo expression time optimization
Procedure


We set the O.D value to 0.1 and cultured 1.5ml cell in 96 deep well plate. At each time point, we transferred 1ml cell from 96 deep well plate to EP tube and 200λ cell to flat 96 well plate for O.D check.
After cell down in EP tube, we discarded the supernatant and resuspended the pellet in 200λ DMSO. We did sonication to the sample and after another cell down, we took the supernatant for TECAN measurement. Also, we checked absorbance after 3 days incubation and before 3days incubation.
We used TECAN at absorbance 620nm to measure the quantity of indigo production

 Results

0818Fig1



We subtracted absorbance value of pure LB from value of samples and we set all samples to have same amount of cell using O.D value
Graph shown below is indigo intensity per cell. After 3 days incubation, indigo seems to be degraded and all the samples seem to have almost same indigo level.
0818Fig1


We excluded 0hr data from the graph to see teh rising tendency clearly.

Discussion

This data shows that the cell successfully produces indigo. We can use this gene as a reporter of our module. However, we only checked the increase of indigo. It may decrease after prolonged culture. We should do additional experiments to get the peak in the graph and optimize the production time of indigo.
Back to the Calendar
October 19th 2012


　Flip Flop

pAutoIntegrase Alternative sequencing with fragments amplification
Results

2_1019Fig1

Back to the Calendar
October 20th 2012

No Special Event!
Back to the Calendar
October 21st 2012

No Special Event!
Back to the Calendar
October 22nd 2012

No Special Event!
Back to the Calendar
October 23rd 2012

No Special Event!
Back to the Calendar
October 24th 2012


　Flip Flop

OE template preparation
Results

2_1024Fig1

gp47, aiiA, LuxI, bFMO for simple and pAuto

2_1024Fig1

promoter and Inverted one

Back to the Calendar
October 25th 2012

　Flip Flop

pAuto intermediate fragment OE PCR
Results

1025Fig1


OE PCR with new primer
Results

2_1025Fig2

pAutoSimple/inverted

2_1025Fig2

pAuto/inverted

Back to the Calendar
October 26th 2012

No Special Event!
Back to the Calendar
October 27th 2012

No Special Event!
Back to the Calendar
October 28th 2012

No Special Event!
Back to the Calendar
October 29th 2012

No Special Event!
Back to the Calendar
October 30th 2012

No Special Event!
Back to the Calendar
October 31st 2012

No Special Event!
Back to the Calendar


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