Team:Yale/Our Project/Notebook/Week 4

From 2010.igem.org

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Thursday 7/1--Smelly bacteria (yay!)& ongoing ligation efforts
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Thursday 7/1--Smelly bacteria (yay!) & ongoing ligation efforts
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<h4> TSI agar assay </h4>
<h4> TSI agar assay </h4>
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<ul>
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*LE392 and DH5alpha cultures in TSI Agar showed some growth. No black color change was visible, but both slants had a rotten egg scent, so suspected generation of H2S at concentrations too low to visibly trigger color change yet.<br/>
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<li>LE392 and DH5alpha cultures in TSI Agar showed some growth. No black color change was visible, but both slants had a rotten egg scent, so suspected generation of H2S at concentrations too low to visibly trigger color change yet.</li><br/>
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</ul>
<h4>PCR work</h4>
<h4>PCR work</h4>
<ul>
<ul>
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<b>Stockpiling plasmids for later</b> <br/>
<b>Stockpiling plasmids for later</b> <br/>
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* Miniprepped LE392 cultures grown overnight,resulting in two Eppendorfs apiece of the following plasmids: B0015 (double terminator), J23114 (constitutive promoter), pSB74 (thiosulfate reductase), R0011 (IPTG-inducible promoter), and P0312 (lacI needed with R0011). Nanodropping gave the following concentrations:<br/>
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<li>Miniprepped LE392 cultures grown overnight,resulting in two Eppendorfs apiece of the following plasmids: B0015 (double terminator), J23114 (constitutive promoter), pSB74 (thiosulfate reductase), R0011 (IPTG-inducible promoter), and P0312 (lacI needed with R0011). Nanodropping gave the following concentrations:</li><br/>
<table>
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<h4>Results of 6/30 Ligation (Ligation Attempt #1)</h4>
<h4>Results of 6/30 Ligation (Ligation Attempt #1)</h4>
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     <li>Started liquid cultures and index plates of four colonies from reactions 2 and 5.</li>
     <li>Started liquid cultures and index plates of four colonies from reactions 2 and 5.</li>
     <li>Also set up colony PCR for all four colonies, spotting the template DNA into tubes with the following contents and running on thermocycler protocol phs50 </li>  
     <li>Also set up colony PCR for all four colonies, spotting the template DNA into tubes with the following contents and running on thermocycler protocol phs50 </li>  
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<table>
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Component Volume
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Distilled Water 27 μL
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<td>Component</td> <td>Volume</td>
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Phusion 5x Buffer 10 μL
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</tr>
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DMSO 1.5 μL
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<tr>
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10 mM dNTPs 1 μL
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<td>Distilled Water</td> <td>27 μL</td>
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10 uM phsABC_F primer 5 μL
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</tr>
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10 uM phsABC_R primer 5 μL
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<tr>
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Phusion Polymerase 0.5 μL
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<td>Phusion 5x Buffer</td> <td>10 μL</td>
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Template DNA spotting
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</tr>
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Total 50 μL
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<td>DMSO</td> <td>1.5 μL</td>
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    * After PCR, ran 1.0% agarose gel of colony PCR reaction solutions at 90 V versus a 1 kb ladder. A preliminary visualization showed no visible non-primer DNA except for a band between 3 and 4 kb for the PCR product from colony 1. Based on a later visualization said band appeared to be closer to 3 kb, suggesting the 3.2 kb B0015 vector rather than the 3.6 kb insert. Unfortunately gel was dropped and damaged irreparably before picture could actually be taken.  
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<td>10 mM dNTPs </td> <td>1 μL</td>
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</tr>
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<tr>
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<td>10 uM phsABC_F primer</td> <td>5 μL</td>
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</tr>
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<tr>
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<td>10 uM phsABC_R primer </td> <td>5 μL</td>
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</tr>
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<td>Phusion Polymerase</td> <td>0.5 μL</td>
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</tr>
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<td>Template DNA </td> <td>spotting</td>
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</tr>
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<td>Total</td> <td>50 μL</td>
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</tr>
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</table>
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<li>After PCR, ran 1.0% agarose gel of colony PCR reaction solutions at 90 V versus a 1 kb ladder. A preliminary visualization showed no visible non-primer DNA except for a band between 3 and 4 kb for the PCR product from colony 1. Based on a later visualization said band appeared to be closer to 3 kb, suggesting the 3.2 kb B0015 vector rather than the 3.6 kb insert. Unfortunately gel was dropped and damaged irreparably before picture could actually be taken. </li>
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</ul>
<i>Wetlab work for this day is also described on pages 37-41 of the hard copy lab notebook </i>
<i>Wetlab work for this day is also described on pages 37-41 of the hard copy lab notebook </i>

Revision as of 11:33, 27 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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  • Fridays short content
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  • Saturday short content
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  • Sunday short content
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