http://2012.igem.org/wiki/index.php?title=Team:XMU-China/protocols&feed=atom&action=historyTeam:XMU-China/protocols - Revision history2024-03-29T08:09:10ZRevision history for this page on the wikiMediaWiki 1.16.0http://2012.igem.org/wiki/index.php?title=Team:XMU-China/protocols&diff=212431&oldid=prevYoubin at 19:36, 26 September 20122012-09-26T19:36:58Z<p></p>
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</table>Youbinhttp://2012.igem.org/wiki/index.php?title=Team:XMU-China/protocols&diff=211276&oldid=prevYoubin: moved Template:Team:XMU-China/protocols to Team:XMU-China/protocols2012-09-26T19:12:05Z<p>moved <a href="/Template:Team:XMU-China/protocols" class="mw-redirect" title="Template:Team:XMU-China/protocols">Template:Team:XMU-China/protocols</a> to <a href="/Team:XMU-China/protocols" title="Team:XMU-China/protocols">Team:XMU-China/protocols</a></p>
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</tr></table>Youbinhttp://2012.igem.org/wiki/index.php?title=Team:XMU-China/protocols&diff=209971&oldid=prevIshuidi at 18:44, 26 September 20122012-09-26T18:44:51Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> - Incubate on ice for 2 min <br /></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> - Incubate on ice for 2 min <br /></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> - Add 790 &mu;L of LB broth to each tube and incubate for an hour at 37 ℃ with shaking. <br /></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> - Add 790 &mu;L of LB broth to each tube and incubate for an hour at 37 ℃ with shaking. <br /></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> - Spread 100 &mu;L and <del class="diffchange diffchange-inline">50uL </del>of each culture on an LB agar plate containing the appropriate antibiotics and incubate overnight at 37 ℃ (spread using beads). </p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> - Spread 100 &mu;L and <ins class="diffchange diffchange-inline">50μL </ins>of each culture on an LB agar plate containing the appropriate antibiotics and incubate overnight at 37 ℃ (spread using beads). </p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p align="left"><strong class="subtitle"><a name="_Toc05" id="Toc4"></a></strong><strong class="subsubtitle">1.4 Plasmid Purification</strong> <br /></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p align="left"><strong class="subtitle"><a name="_Toc05" id="Toc4"></a></strong><strong class="subsubtitle">1.4 Plasmid Purification</strong> <br /></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <strong>- </strong>Centrifuge sample in eppendorf tube approximately 1.5 mL at a time, draining off supernatant after each spin and adding more cell solution <br /></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <strong>- </strong>Centrifuge sample in eppendorf tube approximately 1.5 mL at a time, draining off supernatant after each spin and adding more cell solution <br /></div></td></tr>
</table>Ishuidihttp://2012.igem.org/wiki/index.php?title=Team:XMU-China/protocols&diff=208294&oldid=prevYoubin at 18:10, 26 September 20122012-09-26T18:10:18Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> - Discard the column and store the purified plasmid DNA at -20 &deg;C.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> - Discard the column and store the purified plasmid DNA at -20 &deg;C.</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p align="left" class="subsubtitle"><strong class="subtitle"><a name="_Toc06" id="Toc5"></a></strong><strong>1.5 Reaction system of restriction endonuclease</strong></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p align="left" class="subsubtitle"><strong class="subtitle"><a name="_Toc06" id="Toc5"></a></strong><strong>1.5 Reaction system of restriction endonuclease</strong></p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <p align="left"><img src="https://static.igem.org/mediawiki/2012/7/72/The_digestion_system.jpg" alt="" width="<del class="diffchange diffchange-inline">553" height="297</del>" /><img src="image005.jpg" alt="" width="900" height="1" />- System1、2、3 and 4 are used for Standard BioBrick Assembly .<br /></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <p align="left"><img src="https://static.igem.org/mediawiki/2012/7/72/The_digestion_system.jpg" alt="" width="<ins class="diffchange diffchange-inline">800</ins>" /><img src="image005.jpg" alt="" width="900" height="1" />- System1、2、3 and 4 are used for Standard BioBrick Assembly .<br /></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> - System 5 and 6 are used for restriction analysis. Digestion of sample: at least 500 ng DNA / 10 &mu;L volume. Digest for 1 h at 37 &deg;C, afterwards inactivated by adding 10<strong>&times;</strong> loading buffer and standing for 10 min at room temperature. </p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> - System 5 and 6 are used for restriction analysis. Digestion of sample: at least 500 ng DNA / 10 &mu;L volume. Digest for 1 h at 37 &deg;C, afterwards inactivated by adding 10<strong>&times;</strong> loading buffer and standing for 10 min at room temperature. </p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p align="left"><strong class="subtitle"><a name="_Toc07" id="Toc6"></a></strong><strong class="subsubtitle">1.6 Standard BioBrick Assembly</strong><br /></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p align="left"><strong class="subtitle"><a name="_Toc07" id="Toc6"></a></strong><strong class="subsubtitle">1.6 Standard BioBrick Assembly</strong><br /></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <td width="262" valign="top"><p align="left">0.05 mol/L CaCl<sub>2</sub> solution</p></td></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <td width="262" valign="top"><p align="left">0.05 mol/L CaCl<sub>2</sub> solution</p></td></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <td width="306" valign="top"><p align="left">5.55 g CaCl<sub>2</sub>+1000 mL deionized water(121 ℃ autoclaving for <del class="diffchange diffchange-inline">20min) </del></p></td></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <td width="306" valign="top"><p align="left">5.55 g CaCl<sub>2</sub>+1000 mL deionized water(121 ℃ autoclaving for <ins class="diffchange diffchange-inline">20 min) </ins></p></td></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </tr></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </tr></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </table></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </table></div></td></tr>
</table>Youbinhttp://2012.igem.org/wiki/index.php?title=Team:XMU-China/protocols&diff=195243&oldid=prevFanR at 12:32, 26 September 20122012-09-26T12:32:15Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p align="left" class="subsubtitle"><strong class="subtitle"><a name="_Toc06" id="Toc5"></a></strong><strong>1.5 Reaction system of restriction endonuclease</strong></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p align="left" class="subsubtitle"><strong class="subtitle"><a name="_Toc06" id="Toc5"></a></strong><strong>1.5 Reaction system of restriction endonuclease</strong></p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p align="left"><img src="https://static.igem.org/mediawiki/2012/7/72/The_digestion_system.jpg" alt="" width="553" height="297" /><img src="image005.jpg" alt="" width="900" height="1" />- System1、2、3 and 4 are used for Standard BioBrick Assembly .<br /></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p align="left"><img src="https://static.igem.org/mediawiki/2012/7/72/The_digestion_system.jpg" alt="" width="553" height="297" /><img src="image005.jpg" alt="" width="900" height="1" />- System1、2、3 and 4 are used for Standard BioBrick Assembly .<br /></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> - System 5 and 6 are used for restriction analysis. Digestion of sample: at least 500 ng DNA / 10 &<del class="diffchange diffchange-inline">micro</del>;L volume. Digest for 1 h at 37 &deg;C, afterwards inactivated by adding 10<strong>&times;</strong> loading buffer and standing for 10 min at room temperature. </p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> - System 5 and 6 are used for restriction analysis. Digestion of sample: at least 500 ng DNA / 10 &<ins class="diffchange diffchange-inline">mu</ins>;L volume. Digest for 1 h at 37 &deg;C, afterwards inactivated by adding 10<strong>&times;</strong> loading buffer and standing for 10 min at room temperature. </p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p align="left"><strong class="subtitle"><a name="_Toc07" id="Toc6"></a></strong><strong class="subsubtitle">1.6 Standard BioBrick Assembly</strong><br /></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p align="left"><strong class="subtitle"><a name="_Toc07" id="Toc6"></a></strong><strong class="subsubtitle">1.6 Standard BioBrick Assembly</strong><br /></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> - Digestion of insert: 2 &mu;g~5 &mu;g DNA / 100 &mu;L volume, 10<strong>&times;</strong> H buffer, <em>Eco</em>R I, <em>Spe </em>I. Digestion and inactivation. Clean up the insert via gel electrophoresis. When cutting the insert out of the gel, try avoiding staining or exposure to ultraviolet light of the insert. <br /></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> - Digestion of insert: 2 &mu;g~5 &mu;g DNA / 100 &mu;L volume, 10<strong>&times;</strong> H buffer, <em>Eco</em>R I, <em>Spe </em>I. Digestion and inactivation. Clean up the insert via gel electrophoresis. When cutting the insert out of the gel, try avoiding staining or exposure to ultraviolet light of the insert. <br /></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> - Isolation of Plasmid<br /></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> - Isolation of Plasmid<br /></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> - Digest BioBrick,the system of Restriction analysis refer to table1<br /></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> - Digest BioBrick,the system of Restriction analysis refer to table1<br /></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> - Gel electrophoresis:add 2.2 &<del class="diffchange diffchange-inline">micro</del>;L loading buffer to digestion mixture. An agarose concentration is 1 %.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> - Gel electrophoresis:add 2.2 &<ins class="diffchange diffchange-inline">mu</ins>;L loading buffer to digestion mixture. An agarose concentration is 1 %.</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p align="left"><strong class="subtitle"><a name="_Toc11" id="Toc10"></a></strong><strong class="subsubtitle">1.10 Gel Extraction</strong><br /></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p align="left"><strong class="subtitle"><a name="_Toc11" id="Toc10"></a></strong><strong class="subsubtitle">1.10 Gel Extraction</strong><br /></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> - Weigh a 1.5 mL centrifuge tube for each DNA fragment to be isolated and record the weight.<br /></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> - Weigh a 1.5 mL centrifuge tube for each DNA fragment to be isolated and record the weight.<br /></div></td></tr>
</table>FanRhttp://2012.igem.org/wiki/index.php?title=Team:XMU-China/protocols&diff=195188&oldid=prevFanR at 12:30, 26 September 20122012-09-26T12:30:49Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> - Digestion of vector: 2 &mu;g~5 &mu;g DNA / 100 &mu;L volume, 10<strong>&times;</strong> M buffer, <em>Eco</em>R I, <em>Xba </em>I. Digestion and inactivation. Clean up the insert via gel electrophoresis. When cutting the insert out of the gel, try to avoid staining or exposure to ultraviolet light of the insert.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> - Digestion of vector: 2 &mu;g~5 &mu;g DNA / 100 &mu;L volume, 10<strong>&times;</strong> M buffer, <em>Eco</em>R I, <em>Xba </em>I. Digestion and inactivation. Clean up the insert via gel electrophoresis. When cutting the insert out of the gel, try to avoid staining or exposure to ultraviolet light of the insert.</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p align="left"><strong class="subtitle"><a name="_Toc08" id="Toc7"></a></strong><strong class="subsubtitle">1.7 Suffix Insertion </strong><br /></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p align="left"><strong class="subtitle"><a name="_Toc08" id="Toc7"></a></strong><strong class="subsubtitle">1.7 Suffix Insertion </strong><br /></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> - Digestion of insert: 2 &mu;g~5 &mu;g DNA / 100 &<del class="diffchange diffchange-inline">micro</del>;L volume, 10<strong>&times;</strong> M buffer, <em>Xba </em>I, <em>Pst </em>I. Digestion and inactivation. Clean up the insert.<br /></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> - Digestion of insert: 2 &mu;g~5 &mu;g DNA / 100 &<ins class="diffchange diffchange-inline">mu</ins>;L volume, 10<strong>&times;</strong> M buffer, <em>Xba </em>I, <em>Pst </em>I. Digestion and inactivation. Clean up the insert.<br /></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> - Digestion of vector : 2 &mu;g~5 &mu;g DNA / 100 &<del class="diffchange diffchange-inline">micro</del>;L volume, 10<strong>&times;</strong> H buffer, <em>Spe </em>I,<em> Pst </em>I. Digestion and inactivation. Clean up the vector.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> - Digestion of vector : 2 &mu;g~5 &mu;g DNA / 100 &<ins class="diffchange diffchange-inline">mu</ins>;L volume, 10<strong>&times;</strong> H buffer, <em>Spe </em>I,<em> Pst </em>I. Digestion and inactivation. Clean up the vector.</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p align="left"><strong class="subtitle"><a name="_Toc09" id="Toc8"></a></strong><strong class="subsubtitle">1.8 Ligation </strong><br /></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p align="left"><strong class="subtitle"><a name="_Toc09" id="Toc8"></a></strong><strong class="subsubtitle">1.8 Ligation </strong><br /></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> - After digestion and clean-up, the next step is ligation. ligation at 16 ℃ for 4 h or at 4 ℃ for 16 h. Table 2 is the system of ligation. <br /></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> - After digestion and clean-up, the next step is ligation. ligation at 16 ℃ for 4 h or at 4 ℃ for 16 h. Table 2 is the system of ligation. <br /></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <strong>Components</strong></td></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <strong>Components</strong></td></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <td width="284" valign="top"><p align="left">Volume/&<del class="diffchange diffchange-inline">micro</del>;L </p></td></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <td width="284" valign="top"><p align="left">Volume/&<ins class="diffchange diffchange-inline">mu</ins>;L </p></td></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </tr></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </tr></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <tr></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <tr></div></td></tr>
</table>FanRhttp://2012.igem.org/wiki/index.php?title=Team:XMU-China/protocols&diff=194945&oldid=prevFanR at 12:24, 26 September 20122012-09-26T12:24:22Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> - System 5 and 6 are used for restriction analysis. Digestion of sample: at least 500 ng DNA / 10 &micro;L volume. Digest for 1 h at 37 &deg;C, afterwards inactivated by adding 10<strong>&times;</strong> loading buffer and standing for 10 min at room temperature. </p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> - System 5 and 6 are used for restriction analysis. Digestion of sample: at least 500 ng DNA / 10 &micro;L volume. Digest for 1 h at 37 &deg;C, afterwards inactivated by adding 10<strong>&times;</strong> loading buffer and standing for 10 min at room temperature. </p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p align="left"><strong class="subtitle"><a name="_Toc07" id="Toc6"></a></strong><strong class="subsubtitle">1.6 Standard BioBrick Assembly</strong><br /></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p align="left"><strong class="subtitle"><a name="_Toc07" id="Toc6"></a></strong><strong class="subsubtitle">1.6 Standard BioBrick Assembly</strong><br /></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> - Digestion of insert: 2 &mu;g~5 &mu;g DNA / 100 &<del class="diffchange diffchange-inline">micro</del>;L volume, 10<strong>&times;</strong> H buffer, <em>Eco</em>R I, <em>Spe </em>I. Digestion and inactivation. Clean up the insert via gel electrophoresis. When cutting the insert out of the gel, try avoiding staining or exposure to ultraviolet light of the insert. <br /></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> - Digestion of insert: 2 &mu;g~5 &mu;g DNA / 100 &<ins class="diffchange diffchange-inline">mu</ins>;L volume, 10<strong>&times;</strong> H buffer, <em>Eco</em>R I, <em>Spe </em>I. Digestion and inactivation. Clean up the insert via gel electrophoresis. When cutting the insert out of the gel, try avoiding staining or exposure to ultraviolet light of the insert. <br /></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> - Digestion of vector: 2 &mu;g~5 &mu;g DNA / 100 &<del class="diffchange diffchange-inline">micro</del>;L volume, 10<strong>&times;</strong> M buffer, <em>Eco</em>R I, <em>Xba </em>I. Digestion and inactivation. Clean up the insert via gel electrophoresis. When cutting the insert out of the gel, try to avoid staining or exposure to ultraviolet light of the insert.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> - Digestion of vector: 2 &mu;g~5 &mu;g DNA / 100 &<ins class="diffchange diffchange-inline">mu</ins>;L volume, 10<strong>&times;</strong> M buffer, <em>Eco</em>R I, <em>Xba </em>I. Digestion and inactivation. Clean up the insert via gel electrophoresis. When cutting the insert out of the gel, try to avoid staining or exposure to ultraviolet light of the insert.</p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p align="left"><strong class="subtitle"><a name="_Toc08" id="Toc7"></a></strong><strong class="subsubtitle">1.7 Suffix Insertion </strong><br /></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p align="left"><strong class="subtitle"><a name="_Toc08" id="Toc7"></a></strong><strong class="subsubtitle">1.7 Suffix Insertion </strong><br /></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> - Digestion of insert: 2 &mu;g~5 &mu;g DNA / 100 &micro;L volume, 10<strong>&times;</strong> M buffer, <em>Xba </em>I, <em>Pst </em>I. Digestion and inactivation. Clean up the insert.<br /></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> - Digestion of insert: 2 &mu;g~5 &mu;g DNA / 100 &micro;L volume, 10<strong>&times;</strong> M buffer, <em>Xba </em>I, <em>Pst </em>I. Digestion and inactivation. Clean up the insert.<br /></div></td></tr>
</table>FanRhttp://2012.igem.org/wiki/index.php?title=Team:XMU-China/protocols&diff=194881&oldid=prevFanR at 12:22, 26 September 20122012-09-26T12:22:11Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> - Heat pulse tubes in 42 ℃ water bath for 30 seconds. <br /></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> - Heat pulse tubes in 42 ℃ water bath for 30 seconds. <br /></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> - Incubate on ice for 2 min <br /></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> - Incubate on ice for 2 min <br /></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> - Add 790 <del class="diffchange diffchange-inline">uL </del>of LB broth to each tube and incubate for an hour at 37 ℃ with shaking. <br /></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> - Add 790 <ins class="diffchange diffchange-inline">&mu;L </ins>of LB broth to each tube and incubate for an hour at 37 ℃ with shaking. <br /></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> - Spread 100 <del class="diffchange diffchange-inline">uL </del>and 50uL of each culture on an LB agar plate containing the appropriate antibiotics and incubate overnight at 37 ℃ (spread using beads). </p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> - Spread 100 <ins class="diffchange diffchange-inline">&mu;L </ins>and 50uL of each culture on an LB agar plate containing the appropriate antibiotics and incubate overnight at 37 ℃ (spread using beads). </p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p align="left"><strong class="subtitle"><a name="_Toc05" id="Toc4"></a></strong><strong class="subsubtitle">1.4 Plasmid Purification</strong> <br /></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <p align="left"><strong class="subtitle"><a name="_Toc05" id="Toc4"></a></strong><strong class="subsubtitle">1.4 Plasmid Purification</strong> <br /></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <strong>- </strong>Centrifuge sample in eppendorf tube approximately 1.5 mL at a time, draining off supernatant after each spin and adding more cell solution <br /></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <strong>- </strong>Centrifuge sample in eppendorf tube approximately 1.5 mL at a time, draining off supernatant after each spin and adding more cell solution <br /></div></td></tr>
</table>FanRhttp://2012.igem.org/wiki/index.php?title=Team:XMU-China/protocols&diff=187409&oldid=prevMuxin at 06:15, 26 September 20122012-09-26T06:15:53Z<p></p>
<table style="background-color: white; color:black;">
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <strong class="subtitle"><a name="_Toc15" id="Toc14"></a></strong><strong class="sub3title">2.2.1 Preparation of Samples</strong><br /></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <strong class="subtitle"><a name="_Toc15" id="Toc14"></a></strong><strong class="sub3title">2.2.1 Preparation of Samples</strong><br /></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> - The samples to be tested are cultured in the Basal Medium with appropriate antibiotics, and take 200 &mu;L bacteria liquid to determine its OD<sub>600</sub> at appropriate time.<br /></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> - The samples to be tested are cultured in the Basal Medium with appropriate antibiotics, and take 200 &mu;L bacteria liquid to determine its OD<sub>600</sub> at appropriate time.<br /></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> - Dilute or concentrate the next 200 &mu;<del class="diffchange diffchange-inline">l </del>bacteria liquid in order to let the OD<sub>600</sub> equals to 4.0 while the computational formula is the actual OD<sub>600</sub> * 200=2.0 * X, and X presents the total volume of the bacteria liquid after being diluted or concentrated while its unit is &mu;L as well. <br /></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> - Dilute or concentrate the next 200 &mu;<ins class="diffchange diffchange-inline">L </ins>bacteria liquid in order to let the OD<sub>600</sub> equals to 4.0 while the computational formula is the actual OD<sub>600</sub> * 200=2.0 * X, and X presents the total volume of the bacteria liquid after being diluted or concentrated while its unit is &mu;L as well. <br /></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> - Add 30 &mu;L diluted or concentrated liquid into corresponding 1.5 mL centrifugal tubes, then mix up them with 10 &mu;L loading buffer. <br /></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> - Add 30 &mu;L diluted or concentrated liquid into corresponding 1.5 mL centrifugal tubes, then mix up them with 10 &mu;L loading buffer. <br /></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> - Put these centrifugal tubes into metal bath and heat them in 100 ℃ in around 5 to 8 min, then centrifuge them at the speed of 13000 rpm for 5 min, the supernatant is what we need. </p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> - Put these centrifugal tubes into metal bath and heat them in 100 ℃ in around 5 to 8 min, then centrifuge them at the speed of 13000 rpm for 5 min, the supernatant is what we need. </p></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <td width="262" valign="top"><p align="left">Cationic Mixture</p></td></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <td width="262" valign="top"><p align="left">Cationic Mixture</p></td></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <td width="306" valign="top"><p>1.2 g CMC powder(121 ℃ autoclaving for 15 min) <br /></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <td width="306" valign="top"><p>1.2 g CMC powder(121 ℃ autoclaving for 15 min) <br /></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> 50 mL 4% w/v CaCl<sub>2</sub> <del class="diffchange diffchange-inline">solution(121℃ </del>autoclaving for <del class="diffchange diffchange-inline">15min) </del><br /></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> 50 mL 4% w/v CaCl<sub>2</sub> <ins class="diffchange diffchange-inline">solution(121 ℃ </ins>autoclaving for <ins class="diffchange diffchange-inline">15 min) </ins><br /></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> Under sterile condition, add 50mL of sterile distilled water into the CMC powder. Dissolve the CMC and mix it with the 50 mL 4% w/v CaCl<sub>2</sub> solution. </p></td></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> Under sterile condition, add 50mL of sterile distilled water into the CMC powder. Dissolve the CMC and mix it with the 50 mL 4% w/v CaCl<sub>2</sub> solution. </p></td></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </tr></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </tr></div></td></tr>
</table>Muxinhttp://2012.igem.org/wiki/index.php?title=Team:XMU-China/protocols&diff=186189&oldid=prevZhaoMA at 04:36, 26 September 20122012-09-26T04:36:28Z<p></p>
<table style="background-color: white; color:black;">
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<td colspan='2' style="background-color: white; color:black;">Revision as of 04:36, 26 September 2012</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </tr></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </tr></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </table></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </table></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <p align="left">- Centrifuge 10 mL sample at a time for 1 min at 6,000 rpm, <del class="diffchange diffchange-inline">draining </del>off supernatant after each spin. Repeat the procedure until 0.2~1.0 g deposits <del class="diffchange diffchange-inline">is </del>collected.<br /></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <p align="left">- Centrifuge 10 mL sample at a time for 1 min at 6,000 rpm, <ins class="diffchange diffchange-inline">drain </ins>off supernatant after each spin. Repeat the procedure until 0.2~1.0 g deposits <ins class="diffchange diffchange-inline">are </ins>collected.<br /></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> - Resuspend the deposits with sterile water and centrifuge for 1min at 6000 <del class="diffchange diffchange-inline">rpm,draining </del>off supernatant.<br /></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> - Resuspend the deposits with sterile water and centrifuge for 1min at 6000 <ins class="diffchange diffchange-inline">rpm,drain </ins>off supernatant.<br /></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> - Mix the deposits with sterile water in a mass ratio of 1:5.<br /></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> - Mix the deposits with sterile water in a mass ratio of 1:5.<br /></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> - Add equivalent volume of sodium alginate solution, mixing thoroughly.<br /></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> - Add equivalent volume of sodium alginate solution, mixing thoroughly.<br /></div></td></tr>
</table>ZhaoMA