Team:Yale/Our Project/Notebook/Week 4

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<b> pSB74 transformants<b> <br/>
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<b> pSB74 transformants</b> <br/>
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/>
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/>
<h4> PCR amplification of phsABC </h4>
<h4> PCR amplification of phsABC </h4>
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</table>
</table>
<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>
<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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<t
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<b> PCR reaction </b> <br/>
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</ul>
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<li> Added the following to each of three PCR tubes: 10 uL 5x Phusion HF buffer, 27.5 uL water, and 1 uL of dNTPs.  5 uL were added of both the forward and reverse primers (at 10 uM), as was 1 uL of the template DNA. The matching of tube numbers, template DNA, and primers was as follows:</li>
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<table>
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<tr>
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<td> Tube #</td> <td>2</td> <td>3</td> <td> 4</td>
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</tr>
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<tr>
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<td> Template DNA</td> <td>phsC</td> <td>phsAB</td> <td> phsABC</td>
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</tr>
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<tr>
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<td>Primers</td> <td>phsC_F & phsC_R</td> <td>phsABC_F & phsAB_R</td> <td> phsABC_F & phsC_R</td>
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</tr>
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<li> Chilled tubes on ice, then added 0.5 uL of Phusion DNA polymerase and put in thermocycler. </li>
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<li> Prior to designing thermocycler protocol, used oligocalc to get the following Tm values for the primers. </li>
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<table>
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<tr>
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<td> Primer </td> <td>phsABC_R </td><td>phsABC_F </td><td>phsAB_R </td><td>phsC_F </td>
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</tr>
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<tr>
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<td> Tm </td> <td>73˚C </td><td>66˚C</td><td>64˚C</td><td>72˚C </td>
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</tr>
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</table>
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However, these values seem excessively high, so will just start out with an annealing temperature of 55˚C.  Devised the following "phs" Thermocycler Protocol<br/>
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1. Initial Denaturation-- 2 minutes at 98˚C<br/>
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2. Denaturation--15 seconds at 98˚C<br/>
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3. Annealing--15 seconds at 55˚C<br/>
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4. Extension--3 minutes at 72˚C <br/>
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Steps 2-4 are repeated for 25 cycles
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5. Final Extension--10 minutes at 72˚C <br/>
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6. Hold--indefinitely at 4˚C<br/>
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<b> Gel of PCR Products </b> <br/>
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Revision as of 09:41, 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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