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Team:Yale/Our Project/Notebook/Week 4
From 2010.igem.org
our project
lab notebook: week 4 (6/28 -7/4)
- Monday 6/28--PCR amplification of thiosulfate reductase operon phsABC See more/less
- 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
- 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. PCR reaction
- 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:
- Chilled tubes on ice, then added 0.5 uL of Phusion DNA polymerase and put in thermocycler.
- Prior to designing thermocycler protocol, used oligocalc to get the following Tm values for the primers.
- Tuesday short content See more/less
- Wednesday short content See more/less
- Thursday short content See more/less
- Fridays short content See more/less
- Saturday short content See more/less
- Sunday short content See more/less
pSB74 transformants
Extensive colony growth was seen on plated transformations of pSB74 into DH5alpha and LE392 from 6/25 but none was visible on BL21 plate. Continued failure of BL21 transformation suggests that the homemade competent BL21 cells being used are somehow faulty.
Extensive colony growth was seen on plated transformations of pSB74 into DH5alpha and LE392 from 6/25 but none was visible on BL21 plate. Continued failure of BL21 transformation suggests that the homemade competent BL21 cells being used are somehow faulty.
PCR amplification of phsABC
| Primer | phsABC_F | phsABC_R | phsAB_R | phsC_F |
| Initial Amount | 38.56 nm | 46.58 nm | 29.03 nm | 21.94 nm |
| Volume of water | 385.6 uL | 465.8 uL | 290.3 uL | 219.4 uL |
| Tube # | 2 | 3 | 4 | |
| Template DNA | phsC | phsAB | phsABC | |
| Primers | phsC_F & phsC_R | phsABC_F & phsAB_R | phsABC_F & phsC_R |
| Primer | phsABC_R | phsABC_F | phsAB_R | phsC_F |
| Tm | 73˚C | 66˚C | 64˚C | 72˚C |
1. Initial Denaturation-- 2 minutes at 98˚C
2. Denaturation--15 seconds at 98˚C
3. Annealing--15 seconds at 55˚C
4. Extension--3 minutes at 72˚C
Steps 2-4 are repeated for 25 cycles 5. Final Extension--10 minutes at 72˚C
6. Hold--indefinitely at 4˚C
Gel of PCR Products
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