Team:Yale/Our Project/Notebook/Week 4

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Monday 6/28--PCR amplification of thiosulfate reductase operon phsABC
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<b> pSB74 transformants<b> <br/>
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Extensive colony growth was seen on plated transformations of pSB74 into DH5alpha and LE392 from <a href="http://2010.igem.org/Team:Yale/Our_Project/Notebook/Week_3"> 6/25 </a> but none was visible on BL21 plate.  Continued failure of BL21 transformation suggests that the homemade competent BL21 cells being used are somehow faulty.  <br/>
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<h4> PCR amplification of phsABC </h4>
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<li> Prior to PCR, took aliquots of all primer samples and diluted them first to 100 uM as detailed below by the chart and then again to 10 uM </li>
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<td>Primer</td> <td> phsABC_F </td> <td> phsABC_R </td> <td> phsAB_R </td> <td> phsC_F </td>
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<td> Initial Amount </td> <td>38.56 nm</td> <td>46.58 nm</td><td>29.03 nm</td><td>21.94 nm</td>
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<td> Volume of water </td> <td>385.6 uL</td> <td>465.8 uL</td><td>290.3 uL</td><td>219.4 uL</td>
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<li> Similarly, diluted the DNA sample to 10 ng DNA/uL.  Started with 0.5 uL of miniprepped pSB74 (sample a) at 67.3 ng/uL and added 2.865 uL of water to get the desired concentration.</li>
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Revision as of 09:12, 27 October 2010

iGEM Yale

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

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