http://2012.igem.org/wiki/index.php?title=Team:Cambridge/Diary/Week_8&feed=atom&action=historyTeam:Cambridge/Diary/Week 8 - Revision history2024-03-29T07:59:59ZRevision history for this page on the wikiMediaWiki 1.16.0http://2012.igem.org/wiki/index.php?title=Team:Cambridge/Diary/Week_8&diff=298551&oldid=prevOlijme: /* Monday */2012-10-27T03:59:52Z<p><span class="autocomment">Monday</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Tom miniprepped the lux plasmid out of the e. coli and tried to PCR the split backbone again- only half the vector came out. Interestingly, he tried to induce light in some of his overnight cultures of E. coli with the lux plasmid, but found that they don't glow.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Tom miniprepped the lux plasmid out of the e. coli and tried to PCR the split backbone again- only half the vector came out. Interestingly, he tried to induce light in some of his overnight cultures of E. coli with the lux plasmid, but found that they don't glow.</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>In other news, Oli and Paul began thinking about how to tune our system using an outside parameter, for use with our standardized output system. It may be valuable to spend some time modelling such a system in visual GEC<del class="diffchange diffchange-inline">. See what we did '''here ''need to link'''''</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>In other news, Oli and Paul began thinking about how to tune our system using an outside parameter, for use with our standardized output system. It may be valuable to spend some time modelling such a system in visual GEC.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Tuesday===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Tuesday===</div></td></tr>
</table>Olijmehttp://2012.igem.org/wiki/index.php?title=Team:Cambridge/Diary/Week_8&diff=292996&oldid=prevEmmyft at 00:16, 27 October 20122012-10-27T00:16:13Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Third attempt at PCR (even though we might have it already we are running out of the original DNA fragments... so it will be good if we have some more anyway)- not much luck, positive control was non-existent/very faint. Looks like we have to troubleshoot PCR again...</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Third attempt at PCR (even though we might have it already we are running out of the original DNA fragments... so it will be good if we have some more anyway)- not much luck, positive control was non-existent/very faint. Looks like we have to troubleshoot PCR again...</div></td></tr>
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</table>Emmyfthttp://2012.igem.org/wiki/index.php?title=Team:Cambridge/Diary/Week_8&diff=286506&oldid=prevOlijme at 16:42, 26 October 20122012-10-26T16:42:15Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>!align="center"|[[Team:Cambridge/Diary/Week 13|13]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>!align="center"|[[Team:Cambridge/Diary/Week 13|13]]</div></td></tr>
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</table>Olijmehttp://2012.igem.org/wiki/index.php?title=Team:Cambridge/Diary/Week_8&diff=111651&oldid=prevOlijme at 12:18, 13 September 20122012-09-13T12:18:41Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>!align="center"|[[Team:Cambridge/Diary/Week 11|11]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>!align="center"|[[Team:Cambridge/Diary/Week 11|11]]</div></td></tr>
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</table>Olijmehttp://2012.igem.org/wiki/index.php?title=Team:Cambridge/Diary/Week_8&diff=87603&oldid=prevEmmyft at 08:35, 30 August 20122012-08-30T08:35:08Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>!align="center"|[[Team:Cambridge/Diary/Week 8|8]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>!align="center"|[[Team:Cambridge/Diary/Week 8|8]]</div></td></tr>
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</table>Emmyfthttp://2012.igem.org/wiki/index.php?title=Team:Cambridge/Diary/Week_8&diff=74721&oldid=prevEmmyft: /* Sunday */2012-08-19T19:35:51Z<p><span class="autocomment">Sunday</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>RATIOMETRICA!!!! We *might* finally have it. There are two tiny colonies on one of the plates from yesterday- <strike> though we still cannot tell if they are colonies or bubbles</strike> they are DEFINITELY COLONIES. Hopefully more will grow tomorrow.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>RATIOMETRICA!!!! We *might* finally have it. There are two tiny colonies on one of the plates from yesterday- <strike> though we still cannot tell if they are colonies or bubbles</strike> they are DEFINITELY COLONIES. Hopefully more will grow tomorrow.</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Third attempt at PCR (even though we might have it already we are running out of the original DNA fragments... so it will be good if we have some more anyway).</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Third attempt at PCR (even though we might have it already we are running out of the original DNA fragments... so it will be good if we have some more anyway)<ins class="diffchange diffchange-inline">- not much luck, positive control was non-existent/very faint. Looks like we have to troubleshoot PCR again..</ins>.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{Template:Team:Cambridge/CAM_2012_TEMPLATE_FOOT}}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{Template:Team:Cambridge/CAM_2012_TEMPLATE_FOOT}}</div></td></tr>
</table>Emmyfthttp://2012.igem.org/wiki/index.php?title=Team:Cambridge/Diary/Week_8&diff=74719&oldid=prevEmmyft: /* Sunday */2012-08-19T19:35:07Z<p><span class="autocomment">Sunday</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Sunday===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Sunday===</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>RATIOMETRICA!!!! We *might* finally have it. There are two tiny colonies on one of the plates from yesterday- though we still cannot tell if they are colonies or bubbles. <del class="diffchange diffchange-inline">We will be </del>more <del class="diffchange diffchange-inline">sure </del>tomorrow.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>RATIOMETRICA!!!! We *might* finally have it. There are two tiny colonies on one of the plates from yesterday- <ins class="diffchange diffchange-inline"><strike> </ins>though we still cannot tell if they are colonies or bubbles<ins class="diffchange diffchange-inline"></strike> they are DEFINITELY COLONIES</ins>. <ins class="diffchange diffchange-inline">Hopefully </ins>more <ins class="diffchange diffchange-inline">will grow </ins>tomorrow.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Third attempt at PCR (even though we might have it already we are running out of the original DNA fragments... so it will be good if we have some more anyway).</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Third attempt at PCR (even though we might have it already we are running out of the original DNA fragments... so it will be good if we have some more anyway).</div></td></tr>
</table>Emmyfthttp://2012.igem.org/wiki/index.php?title=Team:Cambridge/Diary/Week_8&diff=74626&oldid=prevEmmyft: /* Thursday */2012-08-19T17:10:23Z<p><span class="autocomment">Thursday</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Learning from positive control's success, Paul, Emmy and Andreas went ahead to concentrate the DNA fragments for the fluorescent construct. Paul did minivac, which evaporated some of the liquid from the existing tubes, while Emmy and Andreas worked on re-PCR the fragments from the existing fragments, and using the new gel extraction protocol. Running both solutions at the same time just show how desperate we are... So the minivac-ed fragments are gibsoned together, cells are transformed and plated, and we should know tomorrow if it has worked. The PCR attempt, however, yielded some interesting results: only the fluorescent proteins came out. However we have gotten rather used to the temper of PCR, so we will just try again on Saturday.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Learning from positive control's success, Paul, Emmy and Andreas went ahead to concentrate the DNA fragments for the fluorescent construct. Paul did minivac, which evaporated some of the liquid from the existing tubes, while Emmy and Andreas worked on re-PCR the fragments from the existing fragments, and using the new gel extraction protocol. Running both solutions at the same time just show how desperate we are... So the minivac-ed fragments are gibsoned together, cells are transformed and plated, and we should know tomorrow if it has worked. The PCR attempt, however, yielded some interesting results: only the fluorescent proteins came out. However we have gotten rather used to the temper of PCR, so we will just try again on Saturday.</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>5th attempt on second half of lux vector- and an unsurprising no. Tom designed sub-split primers and they should come in on Monday...</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">Tom performed a restriction digest on the Lux vector miniprep product- and yes, the restriction map was exactly what we expected. This is followed by a </ins>5th attempt on second half of lux vector- and an unsurprising no. Tom designed sub-split primers and they should come in on Monday...</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The X-Gal assay also produced a beautiful Dulux&trade; colourwheel of blues.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The X-Gal assay also produced a beautiful Dulux&trade; colourwheel of blues.</div></td></tr>
</table>Emmyfthttp://2012.igem.org/wiki/index.php?title=Team:Cambridge/Diary/Week_8&diff=74617&oldid=prevEmmyft at 17:05, 19 August 20122012-08-19T17:05:58Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Emmy dropped by the lab on her way home from London to check on the ratiometrica cells- nothing. Not even the controls... but then, she's realized she has made the oldest mistake in the book- plating the positive controls on amp plates. So she replated some of the leftover culture on Kanamycin plates. We will know tomorrow... (perhaps we should engineer cells that grow extra fast for next year's iGEM project)</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Emmy dropped by the lab on her way home from London to check on the ratiometrica cells- nothing. Not even the controls... but then, she's realized she has made the oldest mistake in the book- plating the positive controls on amp plates. So she replated some of the leftover culture on Kanamycin plates. We will know tomorrow... (perhaps we should engineer cells that grow extra fast for next year's iGEM project)</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Saturday===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Saturday===</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Third attempt at PCR (even though we might have it already we are running out of the original DNA fragments... so it will be good if we have some more anyway).</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Third attempt at PCR (even though we might have it already we are running out of the original DNA fragments... so it will be good if we have some more anyway).</div></td></tr>
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</table>Emmyfthttp://2012.igem.org/wiki/index.php?title=Team:Cambridge/Diary/Week_8&diff=74615&oldid=prevEmmyft at 17:05, 19 August 20122012-08-19T17:05:33Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Thursday===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Thursday===</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Successes! The positive control has produced many colonies, now that we used more concentrated DNA. We'll change the protocol to reflect this.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Successes! The positive control has produced many colonies, now that we used more concentrated DNA. We'll change the protocol to reflect this<ins class="diffchange diffchange-inline">. The reduced amount of T5 didn't really help (there were fewer colonies on those plates), so we will stick with our original recipe for Gibson mastermix</ins>.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Learning from positive control's success, Paul, Emmy and Andreas went ahead to concentrate the DNA fragments for the fluorescent construct. Paul did minivac, which evaporated some of the liquid from the existing tubes, while Emmy and Andreas worked on re-PCR the fragments from the existing fragments, and using the new gel extraction protocol. Running both solutions at the same time just show how desperate we are... So the minivac-ed fragments are gibsoned together, cells are transformed and plated, and we should know tomorrow if it has worked. The PCR attempt, however, yielded some interesting results: only the fluorescent proteins came out. However we have gotten rather used to the temper of PCR, so we will just try again on Saturday.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Learning from positive control's success, Paul, Emmy and Andreas went ahead to concentrate the DNA fragments for the fluorescent construct. Paul did minivac, which evaporated some of the liquid from the existing tubes, while Emmy and Andreas worked on re-PCR the fragments from the existing fragments, and using the new gel extraction protocol. Running both solutions at the same time just show how desperate we are... So the minivac-ed fragments are gibsoned together, cells are transformed and plated, and we should know tomorrow if it has worked. The PCR attempt, however, yielded some interesting results: only the fluorescent proteins came out. However we have gotten rather used to the temper of PCR, so we will just try again on Saturday.</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;">===Saturday===</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 replated positive control on kanamycin plates worked- so the Gibson was working, which means it is possible a design problem. More research into the Gibson protocol tells us we should really not be using Gibson for any fragments shorter than 250bp in the danger of the T5 exo chewing the entire fragments away... which we have two. We will try to tackle the problem by putting in a smaller amount of T5 exo, or/and putting them in later (when the reaction mixture is very close to 50 degrees). Also, we found out that we should use 20x insert to vector. With all these changes, Emmy tried Gibson again with the minivac concentrated DNA. We will know if that has worked tomorrow.</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;">Emmy re-attempted the PCR from Thursday, with increased DMSO concentration, slightly lower annealing temperature, and some of our new polymerase (velocity from bioline)... no bands at all, not even the positive control. It is probably a problem with the mastermix (see Lab book for details). And on a second look at the gel photos from Thursday- there might actually be some other products, but were mistaken for primer dimers. But nevermind- we will try again tomorrow...</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;">RATIOMETRICA!!!! We *might* finally have it. There are two tiny colonies on one of the plates from yesterday- though we still cannot tell if they are colonies or bubbles. We will be more sure tomorrow.</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;">Third attempt at PCR (even though we might have it already we are running out of the original DNA fragments... so it will be good if we have some more anyway).</ins></div></td></tr>
</table>Emmyft