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Team:Yale/Our Project/Notebook/Week 4
From 2010.igem.org
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| - | <b> pSB74 transformants<b> <br/> | + | <b> pSB74 transformants</b> <br/> |
Extensive colony growth was seen on plated transformations of pSB74 into DH5alpha and LE392 from <a href="https://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="https://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> | ||
| - | < | + | <b> PCR reaction </b> <br/> |
| - | </ | + | <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> |
| + | <table> | ||
| + | <tr> | ||
| + | <td> Tube #</td> <td>2</td> <td>3</td> <td> 4</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> Template DNA</td> <td>phsC</td> <td>phsAB</td> <td> phsABC</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Primers</td> <td>phsC_F & phsC_R</td> <td>phsABC_F & phsAB_R</td> <td> phsABC_F & phsC_R</td> | ||
| + | </tr> | ||
| + | <li> Chilled tubes on ice, then added 0.5 uL of Phusion DNA polymerase and put in thermocycler. </li> | ||
| + | <li> Prior to designing thermocycler protocol, used oligocalc to get the following Tm values for the primers. </li> | ||
| + | <table> | ||
| + | <tr> | ||
| + | <td> Primer </td> <td>phsABC_R </td><td>phsABC_F </td><td>phsAB_R </td><td>phsC_F </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td> Tm </td> <td>73˚C </td><td>66˚C</td><td>64˚C</td><td>72˚C </td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | 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/> | ||
| + | 1. Initial Denaturation-- 2 minutes at 98˚C<br/> | ||
| + | 2. Denaturation--15 seconds at 98˚C<br/> | ||
| + | 3. Annealing--15 seconds at 55˚C<br/> | ||
| + | 4. Extension--3 minutes at 72˚C <br/> | ||
| + | Steps 2-4 are repeated for 25 cycles | ||
| + | 5. Final Extension--10 minutes at 72˚C <br/> | ||
| + | 6. Hold--indefinitely at 4˚C<br/> | ||
| + | <b> Gel of PCR Products </b> <br/> | ||
| + | |||
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Revision as of 09:41, 27 October 2010
our project
lab notebook: week 4 (6/28 -7/4)
- Monday 6/28--PCR amplification of thiosulfate reductase operon phsABC See more/less
