Team:Yale/Our Project/Notebook/Week 5

From 2010.igem.org

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<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></li>
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></li>
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></li>
<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></li>
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<li id="nb"><a href="https://2010.igem.org/Team:Yale/Our Project/Notebook/Week 9">week 9</a></li>
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li>
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li>
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li>
<li><a href="https://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li>
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<img src="https://static.igem.org/mediawiki/2010/0/0a/Yale-ligation-products.jpg" />
<img src="https://static.igem.org/mediawiki/2010/0/0a/Yale-ligation-products.jpg" />
</div>
</div>
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       <div id="caption>Gel of colony PCR from ligation attempt #2 Lane 1: 1 kb ladder, Lanes 3-11:PCR products from colonies 1B-9B, in order</div>
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       <div id="caption">Gel of colony PCR from ligation attempt #2 Lane 1: 1 kb ladder, Lanes 3-11:PCR products from colonies 1B-9B, in order</div>
        
        
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<li>Miniprepped liquid cultures grown from colonies of first ligation attempt (colonies 1A-4A) and second ligation attempt (colonies 1B-9B) according to <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols"> standard miniprep protocol.</a> </li>
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<li>Miniprepped liquid cultures grown from colonies of first ligation attempt (colonies 1A-4A) and second ligation attempt (colonies 1B-9B) according to <a href="https://2010.igem.org/Team:Yale/Our_Project/Protocols"> standard miniprep protocol</a> </li>
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<i>This day's activities are also recorded on page 47 and 48 of the hard copy lab notebook. </i>
<i>This day's activities are also recorded on page 47 and 48 of the hard copy lab notebook. </i>
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<li>The diagnostic 1.0% agarose gel was run at 90 V vs. a 1 kb ladder. Each of the thirteen samples gave one diffuse band around 3 kb and no evidence of the 3.6 kb phsABC fragment, so both ligation attempts #1 and #2 were unsuccessful.</li>
<li>The diagnostic 1.0% agarose gel was run at 90 V vs. a 1 kb ladder. Each of the thirteen samples gave one diffuse band around 3 kb and no evidence of the 3.6 kb phsABC fragment, so both ligation attempts #1 and #2 were unsuccessful.</li>
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       Gel of XbaI and SpeI double digest of plasmids from ligation attempts #1 and #2 Lane 1: 1 kb ladder, Lanes 2-5:1A-4A, Lanes 6-14: 1B-9B <br/>
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<div align="center">
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<img src="https://static.igem.org/mediawiki/2010/9/93/Yale-digest-gel.jpg" />
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</div>
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       <div id="caption">Gel of XbaI and SpeI double digest of plasmids from ligation attempts #1 and #2 Lane 1: 1 kb ladder, Lanes 2-5:1A-4A, Lanes 6-14: 1B-9B </div>
        
        
  <li>During digestion prepped plasmid samples for sequencing. Each sample contained 3 μL of plasmid soln (average concentration of 96 ng/μL), 2 μL of 4 mM VF2 (forward primer), and 13 μL of distilled water. Intended to follow up with reverse sequencing of more promising samples, but gel results made it a moot point. </li>
  <li>During digestion prepped plasmid samples for sequencing. Each sample contained 3 μL of plasmid soln (average concentration of 96 ng/μL), 2 μL of 4 mM VF2 (forward primer), and 13 μL of distilled water. Intended to follow up with reverse sequencing of more promising samples, but gel results made it a moot point. </li>
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<ul>
<ul>
<li>Needed more phsABC, so ran five PCR reactions to amplify it from plasmid pSB74. Reaction mixture contents were the same as the DMSO variant used on <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_4">6/30 </a>as was the thermocycler protocol (phs50). The results were then run on a gel</li>
<li>Needed more phsABC, so ran five PCR reactions to amplify it from plasmid pSB74. Reaction mixture contents were the same as the DMSO variant used on <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_4">6/30 </a>as was the thermocycler protocol (phs50). The results were then run on a gel</li>
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       Gel of PCR amplified phsABC Extreme right lane:1 kb ladder, all other lanes PCR reaction mixtures and their spillover
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<div align="center">
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<img src="https://static.igem.org/mediawiki/2010/a/ac/Yale-pcr-gel.jpg" />
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</div>
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       <div id="caption">Gel of PCR amplified phsABC Extreme right lane:1 kb ladder, all other lanes PCR reaction mixtures and their spillover</div>
        
        
<li>Needed chloramphenicol plates for pSB1C3-based constructs, so mixed 350 μL of 50 μg/μL chloramphenicol with an autoclaved solution of 7.5 g agar and 12.5 g LB in 500 mL H2O and poured plates. </li>
<li>Needed chloramphenicol plates for pSB1C3-based constructs, so mixed 350 μL of 50 μg/μL chloramphenicol with an autoclaved solution of 7.5 g agar and 12.5 g LB in 500 mL H2O and poured plates. </li>
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<li>Based on the results, all enzymes appeared to work, as they successfully created a linearized 3.2 kb fragment.</li>
<li>Based on the results, all enzymes appeared to work, as they successfully created a linearized 3.2 kb fragment.</li>
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<div align="center">
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<img src="https://static.igem.org/mediawiki/2010/9/93/Yale-enzyme-test.jpg" />
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</div>
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<i>Wetlab work for this day is also recorded on pages 54-57 of the hard copy lab notebook. </i>
<i>Wetlab work for this day is also recorded on pages 54-57 of the hard copy lab notebook. </i>
</ul>
</ul>

Latest revision as of 02:51, 28 October 2010

iGEM Yale

lab notebook: week 5 (7/5 -7/11)

  • Monday 7/5--Colony PCR of Ligation Attempt #2 Transformants
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  • Tuesday 7/6--Analysis of Results of Ligation Attempts 1 & 2
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  • Wednesday 7/7--Triple ligation attempt--promoter + thiosulfate reductase + terminator
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  • Thursday 7/8--Further ligation efforts
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  • Friday 7/9--Ligation attempt #4 to join phsABC and B0015 terminator.
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  • Saturday 7/10--Transformants from ligation attempts
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  • Sunday 7/11--Prep for copper growth assays of transformants
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