User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/08/29

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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).
    1. 66 °C - 5.9 nM (2%)
    2. 68 °C - 10.4 nM (20%)
    3. 70 °C - 19.3 nM (15%)
    4. 72 °C - 49.4 nM (7%)
    5. 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:
    1. 95 °C 2 min
    2. 95 °C 20 s
    3. X °C 20 s, where X = 54, 56, 58, 60, 62
    4. 72 °C 2 min (this is suboptimal on purpose, in order to get though the experiment more quickly)
    5. Repeat 2-4 an additional 29 times = 30 cycles
    6. 12 °C hold