http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Project/Labwork/MoreProtocols&feed=atom&action=historyTeam:Newcastle/Project/Labwork/MoreProtocols - Revision history2024-03-28T18:17:00ZRevision history for this page on the wikiMediaWiki 1.16.5http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Project/Labwork/MoreProtocols&diff=91040&oldid=prevJanehsy: /* Recovery of cwlD spores */2009-10-10T17:12:21Z<p><span class="autocomment">Recovery of cwlD spores</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Sheffield graciously gave us some non-germinating spores. There are two methods which can be used to recover the cwlD spores, however, neither give complete restoration of germination.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Sheffield graciously gave us some non-germinating spores. There are two methods which can be used to recover the cwlD spores, however, neither give complete restoration of germination.</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">=</del>=== Method A<del class="diffchange diffchange-inline">=</del>===</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>===Method A===</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>; Method A (fast method resulting in partial germination ~ approx. 0.1% recovery)</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>; Method A (fast method resulting in partial germination ~ approx. 0.1% recovery)</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">=</del>=== Method B <del class="diffchange diffchange-inline">=</del>===</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>===Method B===</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>; Method B (slow method involving stripping of spore coat layers for improved germination ~ approx. 10% recovery)</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>; Method B (slow method involving stripping of spore coat layers for improved germination ~ approx. 10% recovery)</div></td></tr>
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</table>Janehsyhttp://2009.igem.org/wiki/index.php?title=Team:Newcastle/Project/Labwork/MoreProtocols&diff=91039&oldid=prevJanehsy: /* Method A */2009-10-10T17:11:43Z<p><span class="autocomment">Method A</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* Heat activated at 70<sup>o</sup>C for 30 min.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* Heat activated at 70<sup>o</sup>C for 30 min.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* Germination in 10 mM L-alanine at 37<sup>o</sup>C.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* Germination in 10 mM L-alanine at 37<sup>o</sup>C.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>; In addition to the protocol, it is important to take note of the following points:</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>; In addition to the protocol, it is important to take note of the following points:</div></td></tr>
</table>Janehsyhttp://2009.igem.org/wiki/index.php?title=Team:Newcastle/Project/Labwork/MoreProtocols&diff=91037&oldid=prevJanehsy: /* Method A */2009-10-10T17:10:41Z<p><span class="autocomment">Method A</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* After the addition of L-alanine to the solution (which would initiate germination), the solution should be left in the incubator for 10 minutes.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* After the addition of L-alanine to the solution (which would initiate germination), the solution should be left in the incubator for 10 minutes.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* After 10 minutes, the eppendorf tube containing the solution should be spinned down for approximately a minute.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* After 10 minutes, the eppendorf tube containing the solution should be spinned down for approximately a minute.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"> </del>* Note: Another eppendorf tube containing water should be placed on the opposite site to balance out the weight. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">*</ins>* Note: Another eppendorf tube containing water should be placed on the opposite site to balance out the weight. </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* After spinning down the solution, the supernatant should be removed and the spores should be resuspended in 1000ul of LB.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* After spinning down the solution, the supernatant should be removed and the spores should be resuspended in 1000ul of LB.</div></td></tr>
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</table>Janehsyhttp://2009.igem.org/wiki/index.php?title=Team:Newcastle/Project/Labwork/MoreProtocols&diff=91036&oldid=prevJanehsy: /* Recovery of cwlD spores */2009-10-10T17:09:32Z<p><span class="autocomment">Recovery of cwlD spores</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* Heat activated at 70<sup>o</sup>C for 30 min.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* Heat activated at 70<sup>o</sup>C for 30 min.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* Germination in 10 mM L-alanine at 37<sup>o</sup>C.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* Germination in 10 mM L-alanine at 37<sup>o</sup>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;">; In addition to the protocol, it is important to take note of the following points:</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;">* Typically, 10ul of cwlD spores are added to the lysozyme and buffer 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;">* After the addition of L-alanine to the solution (which would initiate germination), the solution should be left in the incubator for 10 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 style="color: red; font-weight: bold; text-decoration: none;">* After 10 minutes, the eppendorf tube containing the solution should be spinned down for approximately a minute.</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;"> * Note: Another eppendorf tube containing water should be placed on the opposite site to balance out the weight. </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;">* After spinning down the solution, the supernatant should be removed and the spores should be resuspended in 1000ul of LB.</ins></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>: ''(Image missing)''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>: ''(Image missing)''</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* Germination in 10 mM L-alanine, 50 mM KCl.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* Germination in 10 mM L-alanine, 50 mM KCl.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Midiprep==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Midiprep==</div></td></tr>
</table>Janehsyhttp://2009.igem.org/wiki/index.php?title=Team:Newcastle/Project/Labwork/MoreProtocols&diff=46776&oldid=prevJanehsy: /* Method A */2009-08-14T15:38:34Z<p><span class="autocomment">Method A</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Sekiguchi, J., Akeo, K., Yamamoto, H., Khasanov, F., Alonso, J. & Kuroda, A. (1995). Nucleotide sequence and regulation of a new putative cell wall hydrolase gene, cwlD, which affects germination in ''Bacillus subtilis''. J Bact 19; 5582-5589.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Sekiguchi, J., Akeo, K., Yamamoto, H., Khasanov, F., Alonso, J. & Kuroda, A. (1995). Nucleotide sequence and regulation of a new putative cell wall hydrolase gene, cwlD, which affects germination in ''Bacillus subtilis''. J Bact 19; 5582-5589.</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>* Spores suspended in lysozyme solution (200 g lysozyme per ml <del class="diffchange diffchange-inline">in10 </del>mM potassium phosphate, 50 mM KCl, 1mM <del class="diffchange diffchange-inline">MgCl2</del>).</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>* Spores suspended in lysozyme solution (200 g lysozyme per ml <ins class="diffchange diffchange-inline">in 10 </ins>mM potassium phosphate, 50 mM KCl, 1mM <ins class="diffchange diffchange-inline">MgCl<sub>2</sub></ins>).</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>* Incubation at 37 <del class="diffchange diffchange-inline">oC </del>for 30 min.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>* Incubation at 37<ins class="diffchange diffchange-inline"><sup>o</sup>C </ins>for 30 min.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>* Heat activated at 70 <del class="diffchange diffchange-inline">oC </del>for 30 min.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>* Heat activated at 70<ins class="diffchange diffchange-inline"><sup>o</sup>C </ins>for 30 min.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>* Germination in 10 mM L-alanine at 37 <del class="diffchange diffchange-inline">oC</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>* Germination in 10 mM L-alanine at 37<ins class="diffchange diffchange-inline"><sup>o</sup>C</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>: ''(Image missing)''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>: ''(Image missing)''</div></td></tr>
</table>Janehsyhttp://2009.igem.org/wiki/index.php?title=Team:Newcastle/Project/Labwork/MoreProtocols&diff=45088&oldid=prevGoksel: /* Lambda DNA/Hind III Digest */2009-08-13T10:19:49Z<p><span class="autocomment">Lambda DNA/Hind III Digest</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Lambda DNA/Hind III Digest==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Lambda DNA/Hind III Digest==</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>[http://www.imbb.forth.gr/groups/minotech-new/pdf/lambda_DNA_HindIII_digest.pdf]</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>[http://www.imbb.forth.gr/groups/minotech-new/pdf/lambda_DNA_HindIII_digest.pdf <ins class="diffchange diffchange-inline">Lambda DNA/Hind III Digest</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>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:Newcastle/Footer}}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:Newcastle/Footer}}</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:Newcastle/Right}}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:Newcastle/Right}}</div></td></tr>
</table>Gokselhttp://2009.igem.org/wiki/index.php?title=Team:Newcastle/Project/Labwork/MoreProtocols&diff=45086&oldid=prevGoksel: /* Other Lab Protocols */2009-08-13T10:19:14Z<p><span class="autocomment">Other Lab Protocols</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Inducing competence in JM109 ''E.coli'' cells==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Inducing competence in JM109 ''E.coli'' cells==</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For these ''E.coli'' cells a protocol devised by Promega (the company from which the competence agents were purchased) was used - see [http://www.promega.com/tbs/tb095/tb095.html website] (you will then need to open the Adobe file)</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For these ''E.coli'' cells a protocol devised by Promega (the company from which the competence agents were purchased) was used - see [http://www.promega.com/tbs/tb095/tb095.html website] (you will then need to open the Adobe file)</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;">==Lambda DNA/Hind III Digest==</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;">[http://www.imbb.forth.gr/groups/minotech-new/pdf/lambda_DNA_HindIII_digest.pdf]</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>
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</table>Gokselhttp://2009.igem.org/wiki/index.php?title=Team:Newcastle/Project/Labwork/MoreProtocols&diff=44619&oldid=prevMJR09 at 17:01, 12 August 20092009-08-12T17:01:40Z<p></p>
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<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">__NOTOC__</ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=Other Lab Protocols=</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=Other Lab Protocols=</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>This page contains lab protocols which we have been given by other people. You may also be interested in [[Team:Newcastle/PhilsProtocols|Phil Aldridge's protocols]].</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>This page contains lab protocols which we have been given by other people. You may also be interested in [[Team:Newcastle/PhilsProtocols|Phil Aldridge's protocols]].</div></td></tr>
</table>MJR09http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Project/Labwork/MoreProtocols&diff=44617&oldid=prevMJR09: /* Other Lab Protocols */2009-08-12T17:01:21Z<p><span class="autocomment">Other Lab Protocols</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* [https://2009.igem.org/Team:Newcastle/Project/Labwork/MoreProtocols#Recovery_of_cwlD_spores Recovery of cw1D spores]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* [https://2009.igem.org/Team:Newcastle/Project/Labwork/MoreProtocols#Recovery_of_cwlD_spores Recovery of cw1D spores]</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* [https://2009.igem.org/Team:Newcastle/Project/Labwork/MoreProtocols#Midiprep Conducting a Midi prep]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* [https://2009.igem.org/Team:Newcastle/Project/Labwork/MoreProtocols#Midiprep Conducting a Midi prep]</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>* Inducing competence in JM109 ''E.coli'' cells</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>* <ins class="diffchange diffchange-inline">[https://2009.igem.org/Team:Newcastle/Project/Labwork/MoreProtocols#Inducing_competence_in_JM109_E.coli_cells </ins>Inducing competence in JM109 ''E.coli'' cells<ins class="diffchange diffchange-inline">]</ins></div></td></tr>
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</table>MJR09http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Project/Labwork/MoreProtocols&diff=44614&oldid=prevMJR09 at 17:00, 12 August 20092009-08-12T17:00:51Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Midiprep==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Midiprep==</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We follow the protocol from GenElute for midipreps. ([http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Bulletin/na0200bul.Par.0001.File.tmp/na0200bul.pdf NA0200S_NA0200]). The list of plasmid kits can be accessed from [http://www.sigmaaldrich.com/life-science/molecular-biology/dna-and-rna-purification/hp-plasmid-kits.html Gen Elute's plasmid kits page].</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We follow the protocol from GenElute for midipreps. ([http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Bulletin/na0200bul.Par.0001.File.tmp/na0200bul.pdf NA0200S_NA0200]). The list of plasmid kits can be accessed from [http://www.sigmaaldrich.com/life-science/molecular-biology/dna-and-rna-purification/hp-plasmid-kits.html Gen Elute's plasmid kits page].</div></td></tr>
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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;">==Inducing competence in JM109 ''E.coli'' 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><ins style="color: red; font-weight: bold; text-decoration: none;">For these ''E.coli'' cells a protocol devised by Promega (the company from which the competence agents were purchased) was used - see [http://www.promega.com/tbs/tb095/tb095.html website] (you will then need to open the Adobe file)</ins></div></td></tr>
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