User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/08/25

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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 μMM primer 4 (each) with variable elongation time. Time is is min:sec:
    1. 0:00 - 4.8 nM (1.3%)
    2. 1:00 - 8.6 nM (0.4%)
    3. 2:00 - 20.4 nM (1.0%)
    4. 3:00 - 30.6 nM (0.04%)
    5. 4:00 - 27.8 nM (2%)
    6. 5:00 - 44.3 nM (4%)
    7. 10:00 - 47.9 nM (2%)
  • Obviously, the advice to elongate for 15 s / kb from the Pfu Ultra II DNAP handbook is no good. If I followed that advice, I'd elongate the ΦX174 genome (5386 bp) for 90 s and achieve ~14.5 nM yield, whereas when I elongated for 10 min, I achieveded 47.9 nM, a 3.3-fold increase. Therefore, I will fix the elongation parameter to 2 min / kb and adjust the WP-PCR protocol to elongate for 12 min for ΦX174. I also not that the 4:00 sample seems to be mis-measured, since the rest of the data "looks like it makes sense" when plotted together.
  • Next, testing variable elongation temperature: Te = 66, 68, 70, 72, 74 °C
  • 50 μL WP-PCR:
    • 30.2 μL H2O
    • 6.25 μL 2mM dNTPs (each) (0.25mM each final)
    • 5.5 μL 10X reaction buffer
    • 5.5 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. 58 °C 20 s
    4. X °C 12 min, , where X = 66, 68, 70, 72, 74
    5. Repeat 2-4 an additional 29 times = 30 cycles
    6. 12 °C hold
  • Note, an error was made, as 10X buffer and primers were 10% too concentrated. This shouldn't affect any conclusions about optimal elongation temperature.
  • Publication idea: describe preparation of cell free expression template by WP-PCR. Include WP-PCR optimization, ligation, and gyrase studies. Estimate the mutated population of PCR products using The Polymerase Chain Reaction and Branching Processes by F. Sun.
  • Publication idea: review of cell free gene expression.