Team:Yale/Our Project/Notebook/Week 4

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<li id="nb"><a href="http://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></li>
<li id="nb"><a href="http://2010.igem.org/Team:Yale/Our Project/Notebook/Week 7">week 7</a></li>
<li id="nb"><a href="http://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></li>
<li id="nb"><a href="http://2010.igem.org/Team:Yale/Our Project/Notebook/Week 8">week 8</a></li>
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<li id="nb"><a href="http://2010.igem.org/Team:Yale/Our Project/Notebook/Week 9">week 9</a></li>
<li><a href="http://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li>
<li><a href="http://2010.igem.org/Team:Yale/Our Project/Protocols">protocols</a></li>
<li><a href="http://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li>
<li><a href="http://2010.igem.org/Team:Yale/Our Project/Safety">safety</a></li>
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<h4>PCR amplification of phs </h4>  
<h4>PCR amplification of phs </h4>  
<li> Because the gel run the previous day was no longer good, had to run a second 0.8% agarose gel of the PCR products to determine the success of the PCR reaction. Below is the image of the gel, with a 1 kb ladder in the leftmost lane, phsC in next lane, phsAB in lane 3, and phsABC in lane 4. </li>
<li> Because the gel run the previous day was no longer good, had to run a second 0.8% agarose gel of the PCR products to determine the success of the PCR reaction. Below is the image of the gel, with a 1 kb ladder in the leftmost lane, phsC in next lane, phsAB in lane 3, and phsABC in lane 4. </li>
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The phsC fragment seems to have been amplified with decent success, but phsABC and phAB are only present in trace amounts, so the thermocycler protocol will have to be tweaked. <br/>
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<div align="center">
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<img src="http://2010.igem.org/wiki/images/6/68/Yale-1st-phs-pcr.jpg" />
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</div>
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<div id="caption">
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The phsC fragment seems to have been amplified with decent success, but phsABC and phAB <br /> are only present in trace amounts, so the thermocycler protocol will have to be tweaked. </div> <br />
<li> In the mean time, gel extract the phsC from lane 2 and the phsABC from lane 4 using the microcentrifuge variant of the  <a href="http://2010.igem.org/Team:Yale/Our_Project/Protocols/gel_extract">standard gel extraction protocol </a> </li>
<li> In the mean time, gel extract the phsC from lane 2 and the phsABC from lane 4 using the microcentrifuge variant of the  <a href="http://2010.igem.org/Team:Yale/Our_Project/Protocols/gel_extract">standard gel extraction protocol </a> </li>
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<ul>
<ul>
<li>Ran 1.0% agarose gel of four 6/30 PCR products at 90 V.</li>
<li>Ran 1.0% agarose gel of four 6/30 PCR products at 90 V.</li>
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<div align="center">
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<img src="http://2010.igem.org/wiki/images/8/87/Yale-phs-gel.jpg" />
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</div>
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<div id="caption">
Gel of phsABC and phsAB PCR products or a demonstration of the utility of DMSO Lane 1: 1 kb ladder and 3.6 kb spillover from Lane 2, Lane 2: 3.6 kb phsABC fragment from PCR protocol without DMSO, Lane 4: 2.9 kb phsAB fragment from PCR protocol without DMSO, Lane 6:3.6 kb phsABC fragment from PCR protocol with DMSO, Lane 8: 2.9 kb phsAB fragment from PCR protocol with DMSO
Gel of phsABC and phsAB PCR products or a demonstration of the utility of DMSO Lane 1: 1 kb ladder and 3.6 kb spillover from Lane 2, Lane 2: 3.6 kb phsABC fragment from PCR protocol without DMSO, Lane 4: 2.9 kb phsAB fragment from PCR protocol without DMSO, Lane 6:3.6 kb phsABC fragment from PCR protocol with DMSO, Lane 8: 2.9 kb phsAB fragment from PCR protocol with DMSO
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       Gel of phsABC and phsAB PCR products or a demonstration of the utility of DMSO Lane 1: 1 kb ladder and 3.6 kb spillover from Lane 2, Lane 2: 3.6 kb phsABC fragment from PCR protocol without DMSO, Lane 4: 2.9 kb phsAB fragment from PCR protocol without DMSO, Lane 6:3.6 kb phsABC fragment from PCR protocol with DMSO, Lane 8: 2.9 kb phsAB fragment from PCR protocol with DMSO <br/>
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       Gel of phsABC and phsAB PCR products or a demonstration of the utility of DMSO Lane 1: 1 kb ladder and 3.6 kb spillover from Lane 2, Lane 2: 3.6 kb phsABC fragment from PCR protocol without DMSO, Lane 4: 2.9 kb phsAB fragment from PCR protocol without DMSO, Lane 6:3.6 kb phsABC fragment from PCR protocol with DMSO, Lane 8: 2.9 kb phsAB fragment from PCR protocol with DMSO
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</div>
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<li>Based on gel, it appears that the phs50 thermocycler protocol is an improvement from phs protocol and that including DMSO also improves yield</li>
<li>Based on gel, it appears that the phs50 thermocycler protocol is an improvement from phs protocol and that including DMSO also improves yield</li>
<li>Cut out largest band for each lane and ran through <a href="http://2010.igem.org/Team:Yale/Our_Project/Protocols/gel_extract">standard gel extraction protocol.</a></li>  
<li>Cut out largest band for each lane and ran through <a href="http://2010.igem.org/Team:Yale/Our_Project/Protocols/gel_extract">standard gel extraction protocol.</a></li>  
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<img src="http://2010.igem.org/wiki/images/3/38/Yale-tsi-agar.png" />
<img src="http://2010.igem.org/wiki/images/3/38/Yale-tsi-agar.png" />
</div>
</div>
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       <div id="caption>blurry (apologies!) image of LE392 slant showing FeS precipitate(right) next to un-inoculated slant (left)</div>
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       <div id="caption">blurry (apologies!) image of LE392 slant showing FeS precipitate(right) next to un-inoculated slant (left)</div>
        
        
</ul>
</ul>

Latest revision as of 02:50, 28 October 2010

iGEM Yale

lab notebook: week 4 (6/28 -7/4)

  • Monday 6/28--PCR amplification of thiosulfate reductase operon phsABC
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  • Tuesday 6/29--Gel extraction of PCR amplified phs, TSI agar slant assay for hydrogen sulfide production, & pre-ligation double digestion
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  • Wednesday 6/30--Troubleshooting PCR amplification of thiosulfate reductase gene & continued work toward ligation
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  • Thursday 7/1--Smelly bacteria (yay!) & ongoing ligation efforts
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  • Friday 7/2--Confirmation of hydrogen sulfide production plus more ligation work
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  • Saturday 7/3--More attempts to put a terminator on thiosulfate reductase and a promoter on lacI
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  • Sunday 7/4--Ligation attempt #2 transformants
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