Hypothesis 2: Gene L is necessary for phage propagation.
- Following yesterday's notes, I repeated the whole plasmid PCR protocol for primers 2 (63-mer) and 3 (77-mer) (separate PCRs, since Ta is 63 °C and 63 °C, respectively).
- 60 μL + 10% whole plasmid PCR reaction to test the 63-mer primer pair 2 and the 77-mer primer pair 3.
- 45.87 μL H2O
- 6.6 μL 10X reaction buffer
- 8.25 μL 2mM dNTPs (each) (0.25mM each final)
- 1.32 μL template (0.1nM final)
- 2.64 μL 5 μM each forward/reverse primer mix (200nM each final)
- 1.32 μL PfuUltra II fusion HS DNA polymerase
- I divided the reaction 4×14.1 μL and then made the following 15 μL reactions
- -template, primer 2 (0.3 μL water, 0.6 μL primer 1 mix of 5 μM each S and AS primer 2)
- +template, primer 2 (0.3 μL water, 0.6 μL primer 1 mix of 5 μM each S and AS primer 2)
- -template, primer 3 (0.3 μL water, 0.6 μL primer 4 mix of 5 μM each S and AS primer 3)
- +template, primer 3 (0.3 μL water, 0.6 μL primer 4 mix of 5 μM each S and AS primer 3)
- I then performed whole plasmid PCR with the following cycling parameters:
- 95 °C 2 min
- 95 °C 20 s
- 63 °C 20 s (2) or 66 °C 20 s (3)
- 72 °C 75 s
- Repeat 2-4 an additional 29 times = 30 cycles of PCR
- 72 °C 3 min
- 12 °C hold
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