http://2010.igem.org/wiki/index.php?title=Team:TU_Delft/protocols/transformation&feed=atom&action=historyTeam:TU Delft/protocols/transformation - Revision history2024-03-28T13:30:03ZRevision history for this page on the wikiMediaWiki 1.16.5http://2010.igem.org/wiki/index.php?title=Team:TU_Delft/protocols/transformation&diff=69157&oldid=prevEbrinkman at 09:22, 12 September 20102010-09-12T09:22:52Z<p></p>
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<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 09:22, 12 September 2010</td>
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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">=</del>=Transformation=<del class="diffchange diffchange-inline">=</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>=Transformation=</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>''Materials:''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>''Materials:''</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>- LB medium (warmed to room temperature)</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>- LB medium (warmed to room temperature)</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>- DNA ligation mix</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>- <ins class="diffchange diffchange-inline">Plasmid DNA or </ins>DNA ligation mix</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>- LB plates containing 15-100 μg/mL antibiotic of choice, pre-warmed to 37 °C </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>- LB <ins class="diffchange diffchange-inline">agar </ins>plates containing 15-100 μg/mL antibiotic of choice, pre-warmed to 37 °C </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">Water </del>bath at 37 °C</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>- <ins class="diffchange diffchange-inline">water </ins>bath at 37 °C</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">Shaking </del>incubator at 37 °C. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>- <ins class="diffchange diffchange-inline">shaking </ins>incubator at 37 °C. </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>1. Add 50-100 ng DNA into a 30 μL competent ''E.coli'', and mix gently. Do not mix by pipetting up and down!</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>1. Add 50-100 ng DNA into a 30 μL competent ''E.coli'', and mix gently. Do not mix by pipetting up and down!</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>2. Incubate tube vial on ice for <del class="diffchange diffchange-inline">30 </del>minutes.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>2. Incubate tube vial on ice for <ins class="diffchange diffchange-inline">15 </ins>minutes.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>3. Heat-shocks the cells for exactly 5 minutes at 37 °C without shaking.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>3. Heat-shocks the cells for exactly 5 minutes at 37 °C without shaking.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>4. Immediately transfer the tubes back to ice for 2 minutes.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>4. Immediately transfer the tubes back to ice for 2 minutes.</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>5. Add <del class="diffchange diffchange-inline">250 </del>μL of room temperature LB medium.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>5. Add <ins class="diffchange diffchange-inline">800 </ins>μL of room temperature LB medium.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>6. Cap tube tightly and shake tube horizontally (225 rpm) at 37 °C for 1 hour.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>6. Cap tube tightly and shake tube horizontally (225 rpm) at 37 °C for 1 hour.</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>7<del class="diffchange diffchange-inline">. Plate from each tube 100 μL on an agar plate containing antibiotic</del>. Spin tube, discard supernatant to leave no </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>7. Spin tube <ins class="diffchange diffchange-inline">(2,000 rpm, 4 minutes)</ins>, discard supernatant to leave no more than 100 μL, <ins class="diffchange diffchange-inline">mix </ins>and plate on an agar plate.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>more than 100 μL, <del class="diffchange diffchange-inline">vortex </del>and plate on an agar plate.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>8. Incubate plates overnight at 37 °C.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>8. Incubate plates overnight at 37 °C.</div></td></tr>
</table>Ebrinkmanhttp://2010.igem.org/wiki/index.php?title=Team:TU_Delft/protocols/transformation&diff=15682&oldid=prevEbrinkman: New page: ==Transformation== ''Materials:'' - competent cells - LB medium (warmed to room temperature) - DNA ligation mix - LB plates containing 15-100 μg/mL antibiotic of choice, pre-warmed t...2010-07-05T12:46:38Z<p>New page: ==Transformation== ''Materials:'' - competent cells - LB medium (warmed to room temperature) - DNA ligation mix - LB plates containing 15-100 μg/mL antibiotic of choice, pre-warmed t...</p>
<p><b>New page</b></p><div>==Transformation==<br />
<br />
''Materials:''<br />
<br />
- competent cells<br />
<br />
- LB medium (warmed to room temperature)<br />
<br />
- DNA ligation mix<br />
<br />
- LB plates containing 15-100 μg/mL antibiotic of choice, pre-warmed to 37 °C <br />
<br />
- Water bath at 37 °C<br />
<br />
- Shaking incubator at 37 °C. <br />
<br />
<br />
''Protocol:''<br />
<br />
1. Add 50-100 ng DNA into a 30 μL competent ''E.coli'', and mix gently. Do not mix by pipetting up and down!<br />
<br />
2. Incubate tube vial on ice for 30 minutes.<br />
<br />
3. Heat-shocks the cells for exactly 5 minutes at 37 °C without shaking.<br />
<br />
4. Immediately transfer the tubes back to ice for 2 minutes.<br />
<br />
5. Add 250 μL of room temperature LB medium.<br />
<br />
6. Cap tube tightly and shake tube horizontally (225 rpm) at 37 °C for 1 hour.<br />
<br />
7. Plate from each tube 100 μL on an agar plate containing antibiotic. Spin tube, discard supernatant to leave no <br />
more than 100 μL, vortex and plate on an agar plate.<br />
<br />
8. Incubate plates overnight at 37 °C.</div>Ebrinkman