http://2013.igem.org/wiki/index.php?title=Team:Paris_Saclay/preparation&feed=atom&action=historyTeam:Paris Saclay/preparation - Revision history2024-03-28T19:05:50ZRevision history for this page on the wikiMediaWiki 1.16.5http://2013.igem.org/wiki/index.php?title=Team:Paris_Saclay/preparation&diff=296059&oldid=prevSolenneParisSaclay: /* Preparation of supercompetent E.coli cells */2013-10-04T16:04:02Z<p><span class="autocomment">Preparation of supercompetent E.coli cells</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>6. Centrifuge the solution for 10 min at 4 °C at 3000 rpm (2500g).</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>6. Centrifuge the solution for 10 min at 4 °C at 3000 rpm (2500g).</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>7. Remove and discard supernatant, resuspend the pellet in 80 ml ice-cold Transfo Buffer, incubate on ice for 10 mins.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>7. Remove and discard supernatant, resuspend the pellet in 80 ml ice-cold Transfo Buffer <ins class="diffchange diffchange-inline">(see below)</ins>, incubate on ice for 10 mins.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>8. Centrifuge the solution for 10 min at 4 °C at 3000 rpm (2500g).</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>8. Centrifuge the solution for 10 min at 4 °C at 3000 rpm (2500g).</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>11. Distribute the solution in chilled 1.5ml microcentrifuge tubes (100 μl/tube), frozen in liquid nitrogen and store at -70 ° C.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>11. Distribute the solution in chilled 1.5ml microcentrifuge tubes (100 μl/tube), frozen in liquid nitrogen and store at -70 ° 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;"></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;"></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;"><u>Component of Transfo Buffer:</u> </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;"> HEPES 10 mmol/L</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;"> MnCl2 55 mmol/L</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;"> CaCl2 15 mmol/L</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;"> KCl 250 mmol/L</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;">Preparation of 500 ml of Transfo 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 style="color: red; font-weight: bold; text-decoration: none;">Dissolve 1.19 g HEPES, 1.10 g CaCl2, and 9.32g KCl in 500 ml of water, adjust the ph at 6.7 with KOH, then add 5.44 g MnCl2.</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;">Sterilize the solution by filtration and store the solution at 4°C.</ins></div></td></tr>
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</table>SolenneParisSaclayhttp://2013.igem.org/wiki/index.php?title=Team:Paris_Saclay/preparation&diff=278527&oldid=prevThaonguyenlelam: /* Preparation of supercompetent E.coli cells */2013-10-03T16:19:05Z<p><span class="autocomment">Preparation of supercompetent E.coli cells</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>10. Incubate on ice for 10 min.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>10. Incubate on ice for 10 min.</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>11. Distribute the solution in <del class="diffchange diffchange-inline">150 cold eppendorfs </del>(100 μl/tube), frozen in liquid nitrogen and store at -70 ° C.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>11. Distribute the solution in <ins class="diffchange diffchange-inline">chilled 1.5ml microcentrifuge tubes </ins>(100 μl/tube), frozen in liquid nitrogen and store at -70 ° C.</div></td></tr>
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</table>Thaonguyenlelamhttp://2013.igem.org/wiki/index.php?title=Team:Paris_Saclay/preparation&diff=278501&oldid=prevThaonguyenlelam: /* Preparation of bacteria super competent cells */2013-10-03T16:16:55Z<p><span class="autocomment">Preparation of bacteria super competent cells</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{Team:Paris_Saclay/incl_debut_generique}}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{Team:Paris_Saclay/incl_debut_generique}}</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>= '''Preparation of <del class="diffchange diffchange-inline">bacteria super competent </del>cells''' =</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>= '''Preparation of <ins class="diffchange diffchange-inline">supercompetent ''E.coli'' </ins>cells''' =</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>1. Prepare <del class="diffchange diffchange-inline">2</del>.<del class="diffchange diffchange-inline">5 ml of preculture of bacteria for 24 hours</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>1. Prepare <ins class="diffchange diffchange-inline">the overnight culture of ''E</ins>.<ins class="diffchange diffchange-inline">coli'' strain DH5alphaZ1 at 37C with shaking at 200-250rpm</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>2. In an Erlenmeyer flask of two liters, inoculate <del class="diffchange diffchange-inline">250 ml </del>of LB with <del class="diffchange diffchange-inline">1 ml </del>of <del class="diffchange diffchange-inline">fresh preculture</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>2. In an Erlenmeyer flask of two liters, inoculate <ins class="diffchange diffchange-inline">250ml </ins>of LB with <ins class="diffchange diffchange-inline">2.5ml </ins>of <ins class="diffchange diffchange-inline">overnight culture</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>3. The initial optical density of the solution is measured at t0 by spectrometer.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>3. The initial optical density of the solution is measured at t0 by spectrometer.</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>4. <del class="diffchange diffchange-inline">Let </del>bacteria <del class="diffchange diffchange-inline">cells grow </del>at 20 °C with agitation at <del class="diffchange diffchange-inline">180 </del>rpm <del class="diffchange diffchange-inline">by incubator for 24 h</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>4. <ins class="diffchange diffchange-inline">Grow </ins>bacteria at 20 °C with agitation at <ins class="diffchange diffchange-inline">200-250 </ins>rpm <ins class="diffchange diffchange-inline">to an OD600nm of 0</ins>.<ins class="diffchange diffchange-inline">4 to 0.5</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>5. <del class="diffchange diffchange-inline">The final optical density of </del>the solution <del class="diffchange diffchange-inline">is measured at tfin by spectrometer</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>5. <ins class="diffchange diffchange-inline">Stop </ins>the solution <ins class="diffchange diffchange-inline">on ice (0 °C) for 10 mins</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>6. <del class="diffchange diffchange-inline">Incubate </del>the solution <del class="diffchange diffchange-inline">at 0 °C </del>for 10 min.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>6. <ins class="diffchange diffchange-inline">Centrifuge </ins>the solution for 10 min <ins class="diffchange diffchange-inline">at 4 °C at 3000 rpm (2500g)</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>7. <del class="diffchange diffchange-inline">Centrifuge </del>the <del class="diffchange diffchange-inline">solution </del>for 10 <del class="diffchange diffchange-inline">min at 4 °C and 6000 rpm</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>7. <ins class="diffchange diffchange-inline">Remove and discard supernatant, resuspend </ins>the <ins class="diffchange diffchange-inline">pellet in 80 ml ice-cold Transfo Buffer, incubate on ice </ins>for 10 <ins class="diffchange diffchange-inline">mins</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>8. <del class="diffchange diffchange-inline">Remove and discard supernatant, resuspend </del>the <del class="diffchange diffchange-inline">pellet in 80 ml ice-cold Transfo Buffer, incubate </del>for 10 min at <del class="diffchange diffchange-inline">60 </del>°C.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>8. <ins class="diffchange diffchange-inline">Centrifuge </ins>the <ins class="diffchange diffchange-inline">solution </ins>for 10 min at <ins class="diffchange diffchange-inline">4 </ins>°C <ins class="diffchange diffchange-inline">at 3000 rpm (2500g)</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>9. <del class="diffchange diffchange-inline">Centrifuge </del>the <del class="diffchange diffchange-inline">solution </del>for <del class="diffchange diffchange-inline">10 min at 4 °C and 6000 rpm</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>9. <ins class="diffchange diffchange-inline">Remove and discard supernatant, resuspend </ins>the <ins class="diffchange diffchange-inline">pellet in 20 ml ice-cold Transfo Buffer, add 1 ml DMSO </ins>for <ins class="diffchange diffchange-inline">a final concentration of 7%</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>10. <del class="diffchange diffchange-inline">Remove and discard supernatant, resuspend the pellet in 15 ml </del>ice<del class="diffchange diffchange-inline">-cold Transfo Buffer, add 1 ml DMSO </del>for <del class="diffchange diffchange-inline">a final concentration of 7%</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>10. <ins class="diffchange diffchange-inline">Incubate on </ins>ice for <ins class="diffchange diffchange-inline">10 min</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>11<del class="diffchange diffchange-inline">. Incubate the solution at 0 °C for 10 min.</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>11. Distribute the solution in 150 cold eppendorfs (100 μl/tube), frozen in liquid nitrogen and store at -70 ° C.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">12</del>. Distribute the solution in 150 cold eppendorfs (100 μl/tube), frozen in liquid nitrogen and store at -70 ° C.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
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</table>Thaonguyenlelamhttp://2013.igem.org/wiki/index.php?title=Team:Paris_Saclay/preparation&diff=146876&oldid=prevCarolineMir: Created page with "{{Team:Paris_Saclay/incl_debut_generique}} = '''Preparation of bacteria super competent cells''' = 1. Prepare 2.5 ml of preculture of bacteria for 24 hours. 2. In an Erlenmeyer..."2013-09-22T10:53:38Z<p>Created page with "{{Team:Paris_Saclay/incl_debut_generique}} = '''Preparation of bacteria super competent cells''' = 1. Prepare 2.5 ml of preculture of bacteria for 24 hours. 2. In an Erlenmeyer..."</p>
<p><b>New page</b></p><div>{{Team:Paris_Saclay/incl_debut_generique}}<br />
= '''Preparation of bacteria super competent cells''' =<br />
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1. Prepare 2.5 ml of preculture of bacteria for 24 hours.<br />
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2. In an Erlenmeyer flask of two liters, inoculate 250 ml of LB with 1 ml of fresh preculture.<br />
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3. The initial optical density of the solution is measured at t0 by spectrometer.<br />
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4. Let bacteria cells grow at 20 °C with agitation at 180 rpm by incubator for 24 h.<br />
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5. The final optical density of the solution is measured at tfin by spectrometer.<br />
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6. Incubate the solution at 0 °C for 10 min.<br />
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7. Centrifuge the solution for 10 min at 4 °C and 6000 rpm.<br />
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8. Remove and discard supernatant, resuspend the pellet in 80 ml ice-cold Transfo Buffer, incubate for 10 min at 60 °C.<br />
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9. Centrifuge the solution for 10 min at 4 °C and 6000 rpm.<br />
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10. Remove and discard supernatant, resuspend the pellet in 15 ml ice-cold Transfo Buffer, add 1 ml DMSO for a final concentration of 7%.<br />
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11. Incubate the solution at 0 °C for 10 min.<br />
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12. Distribute the solution in 150 cold eppendorfs (100 μl/tube), frozen in liquid nitrogen and store at -70 ° C.<br />
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{{Team:Paris_Saclay/incl_fin}}</div>CarolineMir