Team:Yale/Our Project/Notebook/Week 4

From 2010.igem.org

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<ul>
<ul>
<li>No visible color change, and decide that it is safe to accelerate growth by moving slants to the 37˚C incubator as research fails to turn up any safety concerns regarding the hydrogen sulfide emitted in this kind of assay. </li>
<li>No visible color change, and decide that it is safe to accelerate growth by moving slants to the 37˚C incubator as research fails to turn up any safety concerns regarding the hydrogen sulfide emitted in this kind of assay. </li>
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</ul>
<b>PCR efforts continue </b><br/>
<b>PCR efforts continue </b><br/>
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<ul>
<li>Redid 6/29 PCR of phsABC and phsAB, but this time ran two trials of each, with and without 3% DMSO, and changed the thermocycler protocol so that the annealing temperature was 50˚C rather than 55˚C, calling the new protocol "phs50."</li>
<li>Redid 6/29 PCR of phsABC and phsAB, but this time ran two trials of each, with and without 3% DMSO, and changed the thermocycler protocol so that the annealing temperature was 50˚C rather than 55˚C, calling the new protocol "phs50."</li>
<li> After running the protocol, found that PCR tubes had popped open and contents had evaporated, so set up PCR a second time to run overnight.</li>
<li> After running the protocol, found that PCR tubes had popped open and contents had evaporated, so set up PCR a second time to run overnight.</li>
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<li>
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Thursday short content
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Thursday 7/1--Smelly bacteria (yay!)& ongoing ligation efforts
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</li>
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Extra content
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<h4> TSI agar assay </h4>
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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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<h4>PCR work</h4>
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<ul>
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<li>Ran 1.0% agarose gel of four 6/30 PCR products at 90 V.</li>
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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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      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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<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>
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<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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</ul>
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<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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<table>
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<tr>
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<td> Sample </td><td> B0015-1</td><td> B0015-2 </td><td>P0312-1</td><td> P0312-1</td><td> J23114-1 </td><td> J23114-2</td><td> pSB74-1</td><td> pSB74-2 </td><td> R0011-1 </td><td> R0011-2 </td>
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</tr>
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<tr>
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<td> Concentration (ng DNA/uL) </td><td> 262.3</td><td> 28569</td><td>129.1</td><td> 92.3</td><td> 189.0 </td><td> 202.6</td><td> 214.4</td><td> 284.4 </td><td> 102.6</td><td> 79.6 </td>
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</tr>
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</table>
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<h4>Results of 6/30 Ligation (Ligation Attempt #1)</h4>
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<ul>
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<li>Plated post-ligation transformants showed the following results: </li>
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Reaction #1 (small control) 0 colonies<br/>
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Reaction #2 (1:1 insert:vector) 2 colonies, numbered 1A and 2A<br/>
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Reaction #3 (2:1 insert:vector) 0 colonies<br/>
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Reaction #4 (large control) 2 colonies<br/>
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Reaction #5 (large 1:1 insert vector) 2 colonies, numbered 3A and 4A<br/>
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Concerned about the small number of colonies and low ratio of number of colonies from actual ligation to number of colonies from control ligation.<br/>
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    <li>Started liquid cultures and index plates of four colonies from reactions 2 and 5.</li>
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    <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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Component Volume
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Distilled Water 27 μL
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Phusion 5x Buffer 10 μL
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DMSO 1.5 μL
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10 mM dNTPs 1 μL
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10 uM phsABC_F primer 5 μL
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10 uM phsABC_R primer 5 μL
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Phusion Polymerase 0.5 μL
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Template DNA spotting
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Total 50 μL
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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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<i>Wetlab work for this day is also described on pages 37-41 of the hard copy lab notebook </i>
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Revision as of 11:27, 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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