http://2008.igem.org/wiki/index.php?title=Template:Team:UC_Berkeley/Notebook/Molly_notes&feed=atom&action=historyTemplate:Team:UC Berkeley/Notebook/Molly notes - Revision history2024-03-28T21:58:10ZRevision history for this page on the wikiMediaWiki 1.16.5http://2008.igem.org/wiki/index.php?title=Template:Team:UC_Berkeley/Notebook/Molly_notes&diff=99960&oldid=prevMolly: /* Molly's Notes */2008-10-30T01:22:11Z<p><span class="autocomment">Molly's Notes</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=== September ===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=== September ===</div></td></tr>
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</table>Mollyhttp://2008.igem.org/wiki/index.php?title=Template:Team:UC_Berkeley/Notebook/Molly_notes&diff=56736&oldid=prevMolly: /* Molly's Notes */2008-10-15T22:54:39Z<p><span class="autocomment">Molly's Notes</span></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>I digested the four minipreps of pK112246 with BglI/XbaI.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>I digested the four minipreps of pK112246 with BglI/XbaI<ins class="diffchange diffchange-inline">. I am expecting band lengths of 1171/5597. It looks as though #1, 2 and 4 worked. I will send out #1 for sequencing of the att sights.</ins></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 class="diffchange diffchange-inline">I also had Jin transform pK112128 #2, pK112245 (the one that was sequenced, #2) and pK112246 #1 into TG1 cells to rescue and plate each on CA</ins>.</div></td></tr>
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</table>Mollyhttp://2008.igem.org/wiki/index.php?title=Template:Team:UC_Berkeley/Notebook/Molly_notes&diff=56732&oldid=prevMolly: /* Molly's Notes */2008-10-15T22:44:34Z<p><span class="autocomment">Molly's Notes</span></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;">I digested the four minipreps of pK112246 with BglI/XbaI.</ins></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>The sequencing data came back for pK112245 and it looks like the attR1 and attR2 sites are both perfect. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The sequencing data came back for pK112245 and it looks like the attR1 and attR2 sites are both perfect. <ins class="diffchange diffchange-inline">Bing mini-prepped my four cultures of pK112246 and I will BglI/XbaI digest them tomorrow. (Thanks Bing!)</ins></div></td></tr>
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</table>Mollyhttp://2008.igem.org/wiki/index.php?title=Template:Team:UC_Berkeley/Notebook/Molly_notes&diff=56459&oldid=prevMolly: /* Molly's Notes */2008-10-14T22:13:22Z<p><span class="autocomment">Molly's Notes</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=== September ===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=== September ===</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;">'''10/14/08''' <br></ins></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;">The sequencing data came back for pK112245 and it looks like the attR1 and attR2 sites are both perfect. </ins></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''10/13/08''' <br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''10/13/08''' <br></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>I miniprepped the five colonies from pK112245 and the one from pK112246 and digested them with BglI/XbaI. If there is no part in it at all, the bands should be 1171/2804. pK112245 is 1171/4985 and pK112246 is 1171/5597.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>I miniprepped the five colonies from pK112245 and the one from pK112246 and digested them with BglI/XbaI. If there is no part in it at all, the bands should be 1171/2804. pK112245 is 1171/4985 and pK112246 is 1171/5597<ins class="diffchange diffchange-inline">. Only colonies 2,4 and 5 from pK112245 looked like they assembled, while pK112246 looked questionable. I sent in colony 2 for sequencing the attR1 and attR2 sites. I will pick the four colonies that grew on my plate from yesterday and miniprep/characterize them tomorrow</ins>.</div></td></tr>
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</table>Mollyhttp://2008.igem.org/wiki/index.php?title=Template:Team:UC_Berkeley/Notebook/Molly_notes&diff=56250&oldid=prevMolly: /* Molly's Notes */2008-10-14T00:08:47Z<p><span class="autocomment">Molly's Notes</span></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;">'''10/13/08''' <br></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;">I miniprepped the five colonies from pK112245 and the one from pK112246 and digested them with BglI/XbaI. If there is no part in it at all, the bands should be 1171/2804. pK112245 is 1171/4985 and pK112246 is 1171/5597.</ins></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;">'''10/12/08''' <br></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;">I came in today and there was only one colony growing for pK112246, and around 10 for pK112245 (of which I picked 5). These are growing up in CA LB in the 30 deg room in TG1 cells. In case none of these are correct, I used the last of the digested materials to try and re-ligate these again. They are on a CA plate in the 30 deg room.</ins></div></td></tr>
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</table>Mollyhttp://2008.igem.org/wiki/index.php?title=Template:Team:UC_Berkeley/Notebook/Molly_notes&diff=55171&oldid=prevMolly: /* September */2008-10-11T23:29:58Z<p><span class="autocomment">September</span></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;">So I've been super busy, but for the first time I got promising results for gateway. So far, I have the two assembly vectors with {prom.xis.int!} and {prom.xis.int.ihfA.ihfB} which are pK112245 and pK112246 respectively, and the two entry vectors again with {prom.xis.int!} and {prom.xis.int.ihfA.ihfB} which are pK112247 and pK112248 respectively. I also have the entry vector for scheme 1 with a fixed attR2 site in it thanks to Christie; it's called pK112128#2. I got sequencing data back this morning telling me that there was still the attR2 mutation in pK112245 and pK112246, so I'm going to take pK112128#2, and PCR it with mea065 and mea066 so I can digest and ligate my {prom.xis.int!} and {prom.xis.int.ihfA.ihfB} parts in. Also, I did an experiment in which I transformed in all the entry vectors directly into MC1061 and plated on CA to make sure they don't have CA resistance. Nothing grew for pBca1256, pK112247, or pK112248 so it looks like its okay! However, I also did this for the assembly vectors pK112128#2, pK112245#4, and pK112246#5, and it seems as though the ccdB in pK112246#5 is really bad since I got a lot of colonies... The other two only yielded about 6 colonies each, meaning the ccdB in these is (for the most part) working in preventing most background from showing up. </ins></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''10/01/08''' <br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''10/01/08''' <br></div></td></tr>
</table>Mollyhttp://2008.igem.org/wiki/index.php?title=Template:Team:UC_Berkeley/Notebook/Molly_notes&diff=50375&oldid=prevMolly: /* September */2008-10-01T20:37:14Z<p><span class="autocomment">September</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=== September ===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=== September ===</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;">'''10/01/08''' <br></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;">Yay! The sequencing showed that the temperature sensitive promoter assembled with K112237 to make K112244! I'll have to wait until late-ish tonight to pick colonies, assuming there will be some to to pick! <crosses fingers></ins></div></td></tr>
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</table>Mollyhttp://2008.igem.org/wiki/index.php?title=Template:Team:UC_Berkeley/Notebook/Molly_notes&diff=49799&oldid=prevMolly: /* September */2008-09-30T23:40:25Z<p><span class="autocomment">September</span></p>
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<td colspan='2' style="background-color: white; color:black;">Revision as of 23:40, 30 September 2008</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=== September ===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=== September ===</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>'''09/30/08''' <br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''09/30/08''' <br></div></td></tr>
</table>Mollyhttp://2008.igem.org/wiki/index.php?title=Template:Team:UC_Berkeley/Notebook/Molly_notes&diff=49798&oldid=prevMolly: /* September */2008-09-30T23:40:03Z<p><span class="autocomment">September</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''09/30/08''' <br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''09/30/08''' <br></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>I ran the two PCRs from yesterday (mea062+mea061 on mv36, and mea062+mea060 on K112244) out on a gel, and the bands looked good. I gel purified these and zymoed these plus the two that worked from yesterday. Then, I digested pBca1256 and pBjh1601CA each with SpeI/MfeI, and mv36 and K112244 each with EcoRI/NheI. After zymoing and eluting with 6.5ul water, I set up ligations between pBca1256+mv36, pBca1256+K112244, pBjh1601CA+mv36, pBjh1601+K112244. Next, I transformed them into TG1 cells, rescued in no-antibiotic LB in the 30 deg room for 1 hour, plated <del class="diffchange diffchange-inline">them </del>on Spec plates which I put in the 30 deg room to let grow.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>I ran the two PCRs from yesterday (mea062+mea061 on mv36, and mea062+mea060 on K112244) out on a gel, and the bands looked good. I gel purified these and zymoed these plus the two that worked from yesterday. Then, I digested pBca1256 and pBjh1601CA each with SpeI/MfeI, and mv36 and K112244 each with EcoRI/NheI. After zymoing and eluting with 6.5ul water, I set up ligations between pBca1256+mv36, pBca1256+K112244, pBjh1601CA+mv36, pBjh1601+K112244. Next, I transformed them into TG1 cells, rescued in no-antibiotic LB in the 30 deg room for 1 hour, plated <ins class="diffchange diffchange-inline">the two with pBca1256 </ins>on Spec <ins class="diffchange diffchange-inline">and the two with pBjh1601CA on CA </ins>plates which I put in the 30 deg room to let grow<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">I also sent out P_ts + K112237 (=K112244) for sequencing with ca998 to make sure the promoter attached in front</ins>.</div></td></tr>
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</table>Mollyhttp://2008.igem.org/wiki/index.php?title=Template:Team:UC_Berkeley/Notebook/Molly_notes&diff=49775&oldid=prevMolly: /* September */2008-09-30T22:55:31Z<p><span class="autocomment">September</span></p>
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<td colspan='2' style="background-color: white; color:black;">Revision as of 22:55, 30 September 2008</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=== September ===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>=== September ===</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;">'''09/30/08''' <br></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;">I ran the two PCRs from yesterday (mea062+mea061 on mv36, and mea062+mea060 on K112244) out on a gel, and the bands looked good. I gel purified these and zymoed these plus the two that worked from yesterday. Then, I digested pBca1256 and pBjh1601CA each with SpeI/MfeI, and mv36 and K112244 each with EcoRI/NheI. After zymoing and eluting with 6.5ul water, I set up ligations between pBca1256+mv36, pBca1256+K112244, pBjh1601CA+mv36, pBjh1601+K112244. Next, I transformed them into TG1 cells, rescued in no-antibiotic LB in the 30 deg room for 1 hour, plated them on Spec plates which I put in the 30 deg room to let grow.</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;"></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;">'''09/29/08''' <br></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;">So over the weekend, Christie did four PCRs for me: mea063+mea064 on pBca1256, mea065+mea066 on pBjh1601CA, mea062+mea061 on mv36, and mea062+mea060 on K112244. However, only the first two appeared to have worked (which I melted in ADB buffer and put in my box), so I set up the second two PCRs over again.</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;"></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;">'''09/25/08''' <br></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;">I ordered oligos mea060-mea066 (NheI-reverse ihfB!, NheI-revers int!, EcoRI-forward pAH57 promoter, SpeI-forward attL1, MfeI-reverse attL1, SpeI-forward attR2, MfeI-reverse attR2, respectively).</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;"></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;">'''09/23/08''' <br></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;">I went ahead and mini-prepped P_ts + K112237 (=K112244). It's in my box, and it's from righty.</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;"></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;">'''09/22/08''' <br></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;">I picked 8 colonies from the {pAH57 promoter}{rbs.xis.int!}{rbs.ihfA!}{rbs.ihfB!} (K112126 + K112237 = K112244) and ran colony PCR on all of them as well as on K112237 alone for control purposes. Only #3 seemed to have assembled, so I grew it up.</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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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''09/21/08''' <br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''09/21/08''' <br></div></td></tr>
</table>Molly