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Team:Cambridge/Protocols/ColonyPCR - Revision history
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Revision history for this page on the wiki
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PM: /* Method */
2010-09-14T15:28:37Z
<p><span class="autocomment">Method</span></p>
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<td colspan='2' style="background-color: white; color:black;">Revision as of 15:28, 14 September 2010</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Method==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Method==</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*Pick a single colony and place in 20µL of H20. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*Pick a single colony and place in 20µL of H20. </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">*Cell lysis:</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">**Incubate 10 min @ 98°C</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">**Freeze 10 min @ -80°C</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">**Vortex for 2-5 min</ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*In a PCR tube mix:</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*In a PCR tube mix:</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>**10µL of 2X Phusion Mastermix</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>**10µL of 2X Phusion Mastermix</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>**1µL of DNA template</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>**1µL of DNA template <ins class="diffchange diffchange-inline">(lysate </ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>**XµL of Primer 1</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>**XµL of Primer 1</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>**XµL of Primer 2</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>**XµL of Primer 2</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>**7µL of Nuclease free H2O</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>**7µL of Nuclease free H2O</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*Run PCR:</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*Run PCR:</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>**Initial Denaturation: <del class="diffchange diffchange-inline">15 min </del>@ 98°C</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>**Initial Denaturation: <ins class="diffchange diffchange-inline">30s </ins>@ 98°C</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>**Denaturation: 10s @ 98°C</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>**Denaturation: 10s @ 98°C</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>**Annealing: 30s @ Y°C</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>**Annealing: 30s @ Y°C</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>**Final Elongation: 10 min @ 72°C</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>**Final Elongation: 10 min @ 72°C</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>**Final Hold: ∞ @ 4°C</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>**Final Hold: ∞ @ 4°C</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Notes==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Notes==</div></td></tr>
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PM
http://2010.igem.org/wiki/index.php?title=Team:Cambridge/Protocols/ColonyPCR&diff=55842&oldid=prev
PM: /* Notes */
2010-08-23T15:07:35Z
<p><span class="autocomment">Notes</span></p>
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<td colspan='2' style="background-color: white; color:black;">Revision as of 15:07, 23 August 2010</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Notes==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Notes==</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>*For Biobrick primers ([http://partsregistry.org/Part:BBa_G00100 VF2] and [http://partsregistry.org/Part:BBa_G00101 <del class="diffchange diffchange-inline">VF</del>] from James Brown we used the following values: </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>*For Biobrick primers ([http://partsregistry.org/Part:BBa_G00100 VF2] and [http://partsregistry.org/Part:BBa_G00101 <ins class="diffchange diffchange-inline">VR</ins>]<ins class="diffchange diffchange-inline">) </ins>from James Brown we used the following values: </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>**X = 1µL</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>**X = 1µL</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>**Y = 65°C</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>**Y = 65°C</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*[http://www.finnzymes.com/pdf/f531_f532_phusion_highfidelity_pcr_mastermix_datasheet_1_9_low.pdf Datasheet for the 2X Phusion Mastermix]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*[http://www.finnzymes.com/pdf/f531_f532_phusion_highfidelity_pcr_mastermix_datasheet_1_9_low.pdf Datasheet for the 2X Phusion Mastermix]</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:Cambridge/Templates/footer}}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:Cambridge/Templates/footer}}</div></td></tr>
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PM
http://2010.igem.org/wiki/index.php?title=Team:Cambridge/Protocols/ColonyPCR&diff=55840&oldid=prev
PM: /* Notes */
2010-08-23T15:06:20Z
<p><span class="autocomment">Notes</span></p>
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<td colspan='2' style="background-color: white; color:black;">Revision as of 15:06, 23 August 2010</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>**X = 1µL</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>**X = 1µL</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>**Y = 65°C</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>**Y = 65°C</div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">*[http://www.finnzymes.com/pdf/f531_f532_phusion_highfidelity_pcr_mastermix_datasheet_1_9_low.pdf Datasheet for the 2X Phusion Mastermix]</ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:Cambridge/Templates/footer}}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:Cambridge/Templates/footer}}</div></td></tr>
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PM
http://2010.igem.org/wiki/index.php?title=Team:Cambridge/Protocols/ColonyPCR&diff=55836&oldid=prev
PM: New page: {{:Team:Cambridge/Templates/header}} {{:Team:Cambridge/Protocol Menu}} =Colony PCR= ==Materials== *2x Phusion Mastermic *Primers *Nuclease free H2O *Cell culture! ==Method== *Pick a sing...
2010-08-23T15:04:14Z
<p>New page: {{:Team:Cambridge/Templates/header}} {{:Team:Cambridge/Protocol Menu}} =Colony PCR= ==Materials== *2x Phusion Mastermic *Primers *Nuclease free H2O *Cell culture! ==Method== *Pick a sing...</p>
<p><b>New page</b></p><div>{{:Team:Cambridge/Templates/header}}<br />
{{:Team:Cambridge/Protocol Menu}}<br />
=Colony PCR=<br />
<br />
==Materials==<br />
*2x Phusion Mastermic<br />
*Primers<br />
*Nuclease free H2O<br />
*Cell culture!<br />
<br />
==Method==<br />
*Pick a single colony and place in 20µL of H20. <br />
*In a PCR tube mix:<br />
**10µL of 2X Phusion Mastermix<br />
**1µL of DNA template<br />
**XµL of Primer 1<br />
**XµL of Primer 2<br />
**7µL of Nuclease free H2O<br />
*Run PCR:<br />
**Initial Denaturation: 15 min @ 98°C<br />
**Denaturation: 10s @ 98°C<br />
**Annealing: 30s @ Y°C<br />
**Elongation: 30s per kb @ 72°C<br />
**Reapeat steps 2-4, 25 to 35 times<br />
**Final Elongation: 10 min @ 72°C<br />
**Final Hold: ∞ @ 4°C<br />
<br />
<br />
==Notes==<br />
*For Biobrick primers ([http://partsregistry.org/Part:BBa_G00100 VF2] and [http://partsregistry.org/Part:BBa_G00101 VF] from James Brown we used the following values: <br />
**X = 1µL<br />
**Y = 65°C<br />
{{:Team:Cambridge/Templates/footer}}</div>
PM