http://2011.igem.org/wiki/index.php?title=Team:Washington/Protocols/Gib_Rxn&feed=atom&action=historyTeam:Washington/Protocols/Gib Rxn - Revision history2024-03-28T16:14:33ZRevision history for this page on the wikiMediaWiki 1.16.0http://2011.igem.org/wiki/index.php?title=Team:Washington/Protocols/Gib_Rxn&diff=236988&oldid=prevSiegeljb: /* Gibson Cloning Protocol */2011-10-13T19:55:44Z<p><span class="autocomment">Gibson Cloning Protocol</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=Gibson Cloning Protocol=</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=Gibson Cloning Protocol=</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Materials Needed==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Materials Needed==</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Phusion DNA polymerase (New England Biolabs)</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Phusion DNA polymerase (New England Biolabs)</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Taq DNA ligase (New England Biolabs)</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Taq DNA ligase (New England Biolabs)</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Equipment==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Equipment==</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">1. </del>Heat block or thermocycler with PCR tubes.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">#</ins>Heat block or thermocycler with PCR tubes.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> </div></td></tr>
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</table>Siegeljbhttp://2011.igem.org/wiki/index.php?title=Team:Washington/Protocols/Gib_Rxn&diff=236987&oldid=prevSiegeljb: /* Gibson Cloning/Assembly */2011-10-13T19:55:21Z<p><span class="autocomment">Gibson Cloning/Assembly</span></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">=Gibson Cloning/Assembly=</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">__NOTOC__</ins></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">*Prepare this mixture on ice to prevent the reaction from beginning early</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">=Gibson Cloning Protocol=</ins></div></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: #ffa; color:black; font-size: smaller;"><div># <del class="diffchange diffchange-inline">Add 15uL of Gibson MasterMix to </del>each <del class="diffchange diffchange-inline">tube</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">==Materials Needed==</ins></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div># <del class="diffchange diffchange-inline">Add ~20</del>-<del class="diffchange diffchange-inline">50 ng </del>of <del class="diffchange diffchange-inline">purified Backbone DNA </del>to the <del class="diffchange diffchange-inline">reaction tube</del>. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>#<ins class="diffchange diffchange-inline">5X isothermal (ISO) reaction buffer (25% PEG-8000, 500 mM Tris-HCl pH 7.5, 50 mM MgCl2, 50 mM DTT, 1 mM </ins>each <ins class="diffchange diffchange-inline">of the 4 dNTPs, and 5 mM NAD). This is prepared as described below</ins>.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div># Add <del class="diffchange diffchange-inline">~</del>20-<del class="diffchange diffchange-inline">50 ng </del>of <del class="diffchange diffchange-inline">purified Insert </del>DNA <del class="diffchange diffchange-inline">to </del>the <del class="diffchange diffchange-inline">reaction tube</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>#<ins class="diffchange diffchange-inline">T5 exonuclease (Epicentre)</ins></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div># <del class="diffchange diffchange-inline">Fill the remaining tubes with </del>ice <del class="diffchange diffchange-inline">cold, distilled H20 </del>to <del class="diffchange diffchange-inline">ensure a final volume of '''20 uL'''</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">#Phusion DNA polymerase (New England Biolabs)</ins></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div># <del class="diffchange diffchange-inline">After making sure all tubes are appropriately labeled</del>, <del class="diffchange diffchange-inline">incubate all samples </del>for <del class="diffchange diffchange-inline">~</del>1 <del class="diffchange diffchange-inline">hour @ 50oC</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">#Taq DNA ligase (New England Biolabs)</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">==Equipment==</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 class="diffchange diffchange-inline">1. Heat block or thermocycler with PCR tubes.</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 class="diffchange diffchange-inline"> </ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">==Procedure==</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 class="diffchange diffchange-inline">#Prepare 5X ISO buffer. Six ml of this buffer can be prepared by combining the following:</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 class="diffchange diffchange-inline">##3 ml of 1 M Tris</ins>-<ins class="diffchange diffchange-inline">HCl pH 7.5</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 class="diffchange diffchange-inline">##150 μl </ins>of <ins class="diffchange diffchange-inline">2 M MgCl2</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 class="diffchange diffchange-inline">##60 μl of 100 mM dGTP</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 class="diffchange diffchange-inline">##60 μl of 100 mM dATP</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 class="diffchange diffchange-inline">##60 μl of 100 mM dTTP</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 class="diffchange diffchange-inline">##60 μl of 100 mM dCTP</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 class="diffchange diffchange-inline">##300 μl of 1 M DTT</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 class="diffchange diffchange-inline">##1.5 g PEG-8000</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 class="diffchange diffchange-inline">##300 μl of 100 mM NAD</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 class="diffchange diffchange-inline">##Add water </ins>to <ins class="diffchange diffchange-inline">6 ml</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 class="diffchange diffchange-inline">##Aliquot 100 μl and store at -20 °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 class="diffchange diffchange-inline">#Prepare an assembly master mixture. This can be prepared by combining </ins>the <ins class="diffchange diffchange-inline">following:</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 class="diffchange diffchange-inline">##320 μl 5X ISO buffer</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 class="diffchange diffchange-inline">##0</ins>.<ins class="diffchange diffchange-inline">64 μl of 10 U/ μl T5 exo</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 class="diffchange diffchange-inline">##20 μl of 2 U/μl Phusion pol</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 class="diffchange diffchange-inline">##160 μl of 40 U/μl Taq lig</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 class="diffchange diffchange-inline">#</ins>#Add <ins class="diffchange diffchange-inline">water to 1.2 ml</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 class="diffchange diffchange-inline">##Aliquot 15 μl and store at -</ins>20 <ins class="diffchange diffchange-inline">°C. This assembly mixture can be stored at </ins>-<ins class="diffchange diffchange-inline">20 °C for at least one year. The enzymes remain active following at least 10 freeze-thaw cycles.</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 class="diffchange diffchange-inline">###This is ideal for the assembly </ins>of DNA <ins class="diffchange diffchange-inline">molecules with 20-150 bp overlaps. For DNA molecules overlapping by larger than 150 bp, prepare </ins>the <ins class="diffchange diffchange-inline">assembly mixture by using 3.2 μl of 10 U/ μl T5 exo</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 class="diffchange diffchange-inline">Thaw a 15 μl assembly mixture aliquot and keep on </ins>ice <ins class="diffchange diffchange-inline">until ready </ins>to <ins class="diffchange diffchange-inline">be used.</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 class="diffchange diffchange-inline">Add 5 μl of DNA to be assembled to the master mixture. The DNA should be in equimolar amounts. Use 10-100 ng of each ~6 kb DNA fragment. For larger DNA segments</ins>, <ins class="diffchange diffchange-inline">increasingly proportionate amounts of DNA should be added (e.g. 250 ng of each 150 kb DNA segment).</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 class="diffchange diffchange-inline">#Incubate at 50 °C </ins>for <ins class="diffchange diffchange-inline">15 to 60 min (60 min is optimal).</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 class="diffchange diffchange-inline">#If cloning is desired, electroporate </ins>1 <ins class="diffchange diffchange-inline">μl of the assembly reaction into 30 μl electrocompetent E</ins>. <ins class="diffchange diffchange-inline">coli.</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">===Based on Methods described in [http://www.nature.com/nmeth/journal/v6/n5/abs/nmeth.1318.html Enzymatic assembly of DNA molecules up to several hundred kilobases], modified by Rob Egbert===</ins></div></td></tr>
</table>Siegeljbhttp://2011.igem.org/wiki/index.php?title=Team:Washington/Protocols/Gib_Rxn&diff=117744&oldid=prevRravic at 04:57, 14 September 20112011-09-14T04:57:21Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Add ~20-50 ng of purified Backbone DNA to the reaction tube. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Add ~20-50 ng of purified Backbone DNA to the reaction tube. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Add ~20-50 ng of purified Insert DNA to the reaction tube.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Add ~20-50 ng of purified Insert DNA to the reaction tube.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div># Fill the remaining tubes with distilled H20 to ensure a final volume of '''20 uL'''</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div># Fill the remaining tubes with <ins class="diffchange diffchange-inline">ice cold, </ins>distilled H20 to ensure a final volume of '''20 uL'''</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># After making sure all tubes are appropriately labeled, incubate all samples for ~1 hour @ 50oC.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># After making sure all tubes are appropriately labeled, incubate all samples for ~1 hour @ 50oC.</div></td></tr>
</table>Rravichttp://2011.igem.org/wiki/index.php?title=Team:Washington/Protocols/Gib_Rxn&diff=117742&oldid=prevRravic at 04:57, 14 September 20112011-09-14T04:57:06Z<p></p>
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<td colspan='2' style="background-color: white; color:black;">Revision as of 04:57, 14 September 2011</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=Gibson Cloning/Assembly=</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=Gibson Cloning/Assembly=</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;"></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;">*Prepare this mixture on ice to prevent the reaction from beginning early</ins></div></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># Add 15uL of Gibson MasterMix to each tube.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Add 15uL of Gibson MasterMix to each tube.</div></td></tr>
</table>Rravichttp://2011.igem.org/wiki/index.php?title=Team:Washington/Protocols/Gib_Rxn&diff=117685&oldid=prevRravic at 04:19, 14 September 20112011-09-14T04:19:18Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Add 15uL of Gibson MasterMix to each tube.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Add 15uL of Gibson MasterMix to each tube.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div># Add ~20-<del class="diffchange diffchange-inline">30 </del>ng of purified Backbone DNA to the reaction tube. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div># Add ~20-<ins class="diffchange diffchange-inline">50 </ins>ng of purified Backbone DNA to the reaction tube. </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div># Add ~20-<del class="diffchange diffchange-inline">30 </del>ng of purified Insert DNA to the reaction tube.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div># Add ~20-<ins class="diffchange diffchange-inline">50 </ins>ng of purified Insert DNA to the reaction tube.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Fill the remaining tubes with distilled H20 to ensure a final volume of '''20 uL'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># Fill the remaining tubes with distilled H20 to ensure a final volume of '''20 uL'''</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># After making sure all tubes are appropriately labeled, incubate all samples for ~1 hour @ 50oC.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div># After making sure all tubes are appropriately labeled, incubate all samples for ~1 hour @ 50oC.</div></td></tr>
</table>Rravichttp://2011.igem.org/wiki/index.php?title=Team:Washington/Protocols/Gib_Rxn&diff=117665&oldid=prevRravic: Created page with "{{Template:Team:Washington/Templates/Top}} =Gibson Cloning/Assembly= # Add 15uL of Gibson MasterMix to each tube. # Add ~20-30 ng of purified Backbone DNA to the reaction tube..."2011-09-14T03:59:51Z<p>Created page with "{{Template:Team:Washington/Templates/Top}} =Gibson Cloning/Assembly= # Add 15uL of Gibson MasterMix to each tube. # Add ~20-30 ng of purified Backbone DNA to the reaction tube..."</p>
<p><b>New page</b></p><div>{{Template:Team:Washington/Templates/Top}}<br />
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=Gibson Cloning/Assembly=<br />
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# Add 15uL of Gibson MasterMix to each tube.<br />
# Add ~20-30 ng of purified Backbone DNA to the reaction tube. <br />
# Add ~20-30 ng of purified Insert DNA to the reaction tube.<br />
# Fill the remaining tubes with distilled H20 to ensure a final volume of '''20 uL'''<br />
# After making sure all tubes are appropriately labeled, incubate all samples for ~1 hour @ 50oC.</div>Rravic