Hypothesis 2: Gene L is necessary for phage propagation.
- Here is the quantiluore results from the WP-PCR of 0.1 nM ΦX174 and 1 μM primer 4 (each) with variable elongation temperature, Te).
- 66 °C - 5.9 nM (2%)
- 68 °C - 10.4 nM (20%)
- 70 °C - 19.3 nM (15%)
- 72 °C - 49.4 nM (7%)
- 74 °C - 43.1 nM (6%)
- Results show maximal PCR at 72 °C, as expected. I recall the only reason I checked this is that the included technical reference for the PfuUltra II Fusion DNAP was incorrect on elongation time.
- Also performed gel electrophoresis on the samples. 0.1 nM ΦX174 showed no visible band, while the PCRed samples showed a distinct band at ~≥ 5 kb linear dsDNA, which is expected, since the samples were 5.4 kb nicked. This is reassuring, since this is the first time I actually viewed good results of ΦX174 WP-PCR.
- Next, testing variable annealing temperature: Ta = 54, 56, 58, 60, 62 °C
- 50 μL WP-PCR:
- 31.2 μL H2O
- 6.25 μL 2mM dNTPs (each) (0.25mM each final)
- 5 μL 10X reaction buffer
- 5 μL 10 μM primer 4 mix
- 1.563 μL 3.2 nM ΦX174 template (0.1 nM final)
- 1 μL PfuUltra II fusion HS DNA polymerase
- Aliquot 5×10μL.
- Cycling parameters:
- 95 °C 2 min
- 95 °C 20 s
- X °C 20 s, where X = 54, 56, 58, 60, 62
- 72 °C 2 min (this is suboptimal on purpose, in order to get though the experiment more quickly)
- Repeat 2-4 an additional 29 times = 30 cycles
- 12 °C hold
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PHIX174 Cell Free Expression
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