http://2010.igem.org/wiki/index.php?title=Team:BCCS-Bristol/Wetlab/standard_protocols&feed=atom&action=historyTeam:BCCS-Bristol/Wetlab/standard protocols - Revision history2024-03-28T19:13:21ZRevision history for this page on the wikiMediaWiki 1.16.5http://2010.igem.org/wiki/index.php?title=Team:BCCS-Bristol/Wetlab/standard_protocols&diff=190651&oldid=prevTtodd at 18:00, 27 October 20102010-10-27T18:00:52Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>This page provides a database of all the standard protocols used throughout our project. Some procedures are directly listed, whilst those provided by commercial kits are simply linked to.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>This page provides a database of all the standard protocols used throughout our project. Some procedures are directly listed, whilst those provided by commercial kits are simply linked to.</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>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Making Competent Cells==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Making Competent Cells==</div></td></tr>
</table>Ttoddhttp://2010.igem.org/wiki/index.php?title=Team:BCCS-Bristol/Wetlab/standard_protocols&diff=190622&oldid=prevTtodd at 18:00, 27 October 20102010-10-27T18:00:03Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=Standard Protocols=</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=Standard Protocols=</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><ins class="diffchange diffchange-inline">{{:Team:BCCS-Bristol/newtoc}}</ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>This page provides a database of all the standard protocols used throughout our project. Some procedures are directly listed, whilst those provided by commercial kits are simply linked to.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>This page provides a database of all the standard protocols used throughout our project. Some procedures are directly listed, whilst those provided by commercial kits are simply linked to.</div></td></tr>
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</table>Ttoddhttp://2010.igem.org/wiki/index.php?title=Team:BCCS-Bristol/Wetlab/standard_protocols&diff=187605&oldid=prevKcoyte at 16:23, 27 October 20102010-10-27T16:23:07Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:BCCS-Bristol/Header}}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:BCCS-Bristol/Header}}</div></td></tr>
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<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;"><center> • [[:Team:BCCS-Bristol/Wetlab/standard_protocols| Standard Protocols]] • [[:Team:BCCS-Bristol/Wetlab/K381001_Construction| BBa_K381001 Construction]] • [[:Team:BCCS-Bristol/Wetlab/K381001_Characterisation| BBa_K381001 Characterisation]] • [[:Team:BCCS-Bristol/Wetlab/signal_soil| Detecting Signals in Soil]] • [[:Team:BCCS-Bristol/Wetlab/making_beads| Making Beads]] • [[:Team:BCCS-Bristol/Wetlab/difference_solution| Beads In Solution]] • </del></div></td><td colspan="2"> </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;"> • [[:Team:BCCS-Bristol/Wetlab/difference_soil| Beads On Soil]] • [[:Team:BCCS-Bristol/Wetlab/characterising_ratio| Signal Calibration]] • [[:Team:BCCS-Bristol/Wetlab/RFPbead_tests| Final Beads]] • </del></div></td><td colspan="2"> </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;"> • [[:Team:BCCS-Bristol/Wetlab/BetaGalactosidaseAssays| Miller Assays ]] • </center> </del></div></td><td colspan="2"> </td></tr>
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</table>Kcoytehttp://2010.igem.org/wiki/index.php?title=Team:BCCS-Bristol/Wetlab/standard_protocols&diff=181444&oldid=prevKcoyte at 12:46, 27 October 20102010-10-27T12:46:45Z<p></p>
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<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;"><center> • [[:Team:BCCS-Bristol/Wetlab/standard_protocols| Standard Protocols]] • [[:Team:BCCS-Bristol/Wetlab/K381001_Construction| BBa_K381001 Construction]] • [[:Team:BCCS-Bristol/Wetlab/K381001_Characterisation| BBa_K381001 Characterisation]] • [[:Team:BCCS-Bristol/Wetlab/signal_soil| Detecting Signals in Soil]] • [[:Team:BCCS-Bristol/Wetlab/making_beads| Making Beads]] • [[:Team:BCCS-Bristol/Wetlab/difference_solution| Beads In Solution]] • </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;"> • [[:Team:BCCS-Bristol/Wetlab/difference_soil| Beads On Soil]] • [[:Team:BCCS-Bristol/Wetlab/characterising_ratio| Signal Calibration]] • [[:Team:BCCS-Bristol/Wetlab/RFPbead_tests| Final Beads]] • </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;"> • [[:Team:BCCS-Bristol/Wetlab/BetaGalactosidaseAssays| Miller Assays ]] • </center> </ins></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=Standard Protocols=</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=Standard Protocols=</div></td></tr>
</table>Kcoytehttp://2010.igem.org/wiki/index.php?title=Team:BCCS-Bristol/Wetlab/standard_protocols&diff=166223&oldid=prevKcoyte at 20:46, 26 October 20102010-10-26T20:46:40Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=Standard Protocols=</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=Standard Protocols=</div></td></tr>
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</table>Kcoytehttp://2010.igem.org/wiki/index.php?title=Team:BCCS-Bristol/Wetlab/standard_protocols&diff=166198&oldid=prevKcoyte at 20:45, 26 October 20102010-10-26T20:45:16Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>This procedure is for the transformation of cells rendered competent by the procedure above. When using NovaBlues a different, quicker method can be followed.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>This procedure is for the transformation of cells rendered competent by the procedure above. When using NovaBlues a different, quicker method can be followed.</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>#Add the DNA to be transformed to an empty, chilled microfuge tube</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div># Add the DNA to be transformed to an empty, chilled microfuge tube</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>#Add 100μl of icecold competent E. coli</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div># Add 100μl of icecold competent E. coli</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>#Incubate on ice for 30mins (or longer)</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div># Incubate on ice for 30mins (or longer)</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>#Heat shock by placing at 42°C got 2 mins</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div># Heat shock by placing at 42°C got 2 mins</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>#Add 0.5ml of Lennox broth and incubate at 37°C for 30-90mins</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div># Add 0.5ml of Lennox broth and incubate at 37°C for 30-90mins</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>#Pellet the cells by spinning briefly in a minifuge. Discard the supernatant</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div># Pellet the cells by spinning briefly in a minifuge. Discard the supernatant</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>#Resuspend each pellet in 100μl of Lennox broth and plate out onto an agar plate containing appropriate antibiotics for plasmid selection. Incubate the plate at 37°C overnight.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div># Resuspend each pellet in 100μl of Lennox broth and plate out onto an agar plate containing appropriate antibiotics for plasmid selection. Incubate the plate at 37°C overnight.</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">==Transforming NovaBlues==</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><ins class="diffchange diffchange-inline">This procedure should only be used when transforming commercial competent cells.</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"># Thaw required number of cell tubes on ice, mix gently to ensure suspension</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 5mL DNA to 50μL cells, stir gently to mix</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"># Place tubes on ice for 5 minutes</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"># Heat shock for 30s at 42°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"># Place tubes on ice for 2 minutes</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 250μL of SOC medium</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"># Plate out 100μL of cell suspension, incubate overnight at 37°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> </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">==Minipreps, Midipreps and Gel Extractions==</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><ins class="diffchange diffchange-inline">Gel extractions, Mini and midipreps were performed using Qiagen kits, following the procedures provided by them:</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">[http://www.qiagen.com/literature/render.aspx?id=370 Miniprep]</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">[http://www.qiagen.com/literature/render.aspx?id=369 Midiprep]</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">[http://www.qiagen.com/literature/render.aspx?id=103715 Gel Extraction]</ins></div></td></tr>
</table>Kcoytehttp://2010.igem.org/wiki/index.php?title=Team:BCCS-Bristol/Wetlab/standard_protocols&diff=166053&oldid=prevKcoyte at 20:35, 26 October 20102010-10-26T20:35:56Z<p></p>
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<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;">=Standard Protocols=</ins></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>This page provides a database of all the standard protocols used throughout our project. Some procedures are directly listed, whilst those provided by commercial kits are simply linked to.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>This page provides a database of all the standard protocols used throughout our project. Some procedures are directly listed, whilst those provided by commercial kits are simply linked to.</div></td></tr>
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</table>Kcoytehttp://2010.igem.org/wiki/index.php?title=Team:BCCS-Bristol/Wetlab/standard_protocols&diff=166043&oldid=prevKcoyte: New page: This page provides a database of all the standard protocols used throughout our project. Some procedures are directly listed, whilst those provided by commercial kits are simply linked to....2010-10-26T20:35:29Z<p>New page: This page provides a database of all the standard protocols used throughout our project. Some procedures are directly listed, whilst those provided by commercial kits are simply linked to....</p>
<p><b>New page</b></p><div>This page provides a database of all the standard protocols used throughout our project. Some procedures are directly listed, whilst those provided by commercial kits are simply linked to.<br />
<br />
==Making Competent Cells==<br />
<br />
<br />
We used this procedure to make both competent ''E. coli'' MG1655 and XL1-blue. Where we were used particularly small amounts of DNA we used commercial NovaBlue cells.<br />
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# Inoculate 50ml Lennox broth in a 150ml flask with 1ml of an overnight culture of E. coli.<br />
# Incubate at 37°C until mid-logarithmic phase (A600 ~0.35, approx 2h (we checked after 1h30))<br />
# Transfer culture to a sterile, chilled, centrifuge tube. Incubate on ice for 10min<br />
# Harvest cells by 5min. centrifugation at 3600rpm at 4°C. Discard supernatant.<br />
# Resuspend cell pellet in 20ml of ice cold 0.1M CaCl2 Incubate on ice for 20min<br />
# Harvest cells by 5min. centrifugation at 3600rpm at 4°C. Discard supernatant.<br />
# Either: Resuspend pellet in 5ml ice cold CaCl2. Incubate on ice for at least 30mins prior to use (competence will continue to increase for up to 24h if cells stored on ice. Or:<br />
# Resuspend pellet in 3ml ice-cold 0.1M CaCl2 plus 2ml ice-cold 50%glycerol. Incubate on ice for 30min then store in aliquots at -80°C.<br />
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<br />
==Transforming Competent Cells==<br />
<br />
<br />
This procedure is for the transformation of cells rendered competent by the procedure above. When using NovaBlues a different, quicker method can be followed.<br />
<br />
#Add the DNA to be transformed to an empty, chilled microfuge tube<br />
#Add 100μl of icecold competent E. coli<br />
#Incubate on ice for 30mins (or longer)<br />
#Heat shock by placing at 42°C got 2 mins<br />
#Add 0.5ml of Lennox broth and incubate at 37°C for 30-90mins<br />
#Pellet the cells by spinning briefly in a minifuge. Discard the supernatant<br />
#Resuspend each pellet in 100μl of Lennox broth and plate out onto an agar plate containing appropriate antibiotics for plasmid selection. Incubate the plate at 37°C overnight.</div>Kcoyte