Team:Yale/Our Project/Notebook/Week 5

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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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<li>Ran colony PCR of colonies 1B-9B from ligation attempt #2 according to the same protocol as used on <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_4"> 7/1</a>.</li>
<li>Ran colony PCR of colonies 1B-9B from ligation attempt #2 according to the same protocol as used on <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_4"> 7/1</a>.</li>
  <li>Then ran a 1.0% agarose gel of the nine PCR reaction solutions against a 1 kb ladder, but the resulting gel failed to show any evidence of either insert or vector, so will have to rely on sequencing and digests of miniprepped plasmids.</li>
  <li>Then ran a 1.0% agarose gel of the nine PCR reaction solutions against a 1 kb ladder, but the resulting gel failed to show any evidence of either insert or vector, so will have to rely on sequencing and digests of miniprepped plasmids.</li>
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       Gel of colony PCR from ligation attempt #2 Lane 1: 1 kb ladder, Lanes 3-11:PCR products from colonies 1B-9B, in order<br/>
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<div align="center">
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<img src="https://static.igem.org/mediawiki/2010/0/0a/Yale-ligation-products.jpg" />
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</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>
</ul>
</ul>
<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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Digestion Reaction Components
Digestion Reaction Components
</td></tr> <tr> <td>
</td></tr> <tr> <td>
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phsABC Digestion </td> <td> B0015 Digestion </td> <td>pSB1C3 Digestion
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phsABC Digestion </td> <td> B0015 Digestion </td> <td>pSB1C3 Digestionhttps://2010.igem.org/Main_Page
</td></tr> <tr> <td>
</td></tr> <tr> <td>
5 μL EcoRI Buffer 10x </td> <td>5 μL NEB Buffer 3 10x </td> <td> 5 μL EcoRI Buffer 10x
5 μL EcoRI Buffer 10x </td> <td>5 μL NEB Buffer 3 10x </td> <td> 5 μL EcoRI Buffer 10x
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Followed with 20 minute heatkill at 80°C.<br/>
Followed with 20 minute heatkill at 80°C.<br/>
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<ul>
<li>Loaded all three enzyme activity tests on a 1.0% agarose gel and ran them at 90 V versus a 1 kb ladder and circular B0015 plasmid. </li>
<li>Loaded all three enzyme activity tests on a 1.0% agarose gel and ran them at 90 V versus a 1 kb ladder and circular B0015 plasmid. </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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</ul>
 
<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>
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</ul>
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<a id="link" href="javascript:ReverseDisplay('saturday')">See more/less</a>
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Sunday 7/11--
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Sunday 7/11--Prep for copper growth assays of transformants
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<h4>
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<b>Ligation work </b> <br/>
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<ul>
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<li> Realize that mistakenly plated triple ligation reaction on ampicillin plate rather than the required chloramphenicol, so none of the colonies present are of value.  Fortunately still have transformation solution, so replate it, this time on a chloramphenicol </li>
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<li> Inoculated overnight liquid cultures of ligation attempt #4 transformants  (#9-#24 on index plates) for miniprep the following morning </li>
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<b>Growth assay prep work </b> <br/>
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<li> Want to see if pSB74 confers any copper resistance on <i>E. coli</i>, since it should help them precipitate nearby copper and thus reduce the copper concentration that they experience. </li>
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<li> Inoculated liquid cultures of  LE392 with pSB74 in ampicillin LB and untransformed LE392 in plain LB and left to grow overnight on shaker at 37˚C for growth assays the following day </li>
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<li> Prepared CuSO<sub>4</sub> solutions in LB.  Each well in the 96-well plate will have 225 uL of copper solution and 25 uL of cell solution, so the copper solution must have a CuSO<sub>4</sub> concentration that is 10/9 the desired final values. The copper solutions must also contain ampicillin and IPTG at the same levels as the cultures that will be introduced. The desired  CuSO<sub>4</sub> concentrations are those used in the wide and narrow concentration range trials on <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook"> 6/10 </a> and <a href="https://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_2"> 6/14 </a>. </li>
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</ul>
<i>Wetlab work for this day is also recorded on pages 59-61 of the hard copy lab notebook.</i>
<i>Wetlab work for this day is also recorded on pages 59-61 of the hard copy lab notebook.</i>
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<!------------- Sunday ------------->

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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