User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/09/17

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Hypothesis 2: Gene L is necessary for phage propagation.

  • Gel results from WP-PCR on 09/14/2012 showed no amplification. I have a new enzyme now, PfuUltra1, so I will do a simple test to see if I can get amplification in the first place.
  • 50 μL WP-PCR reaction:
    • 31.2 μL H2O
    • 6.25 μL 2mM dNTPs (each) (0.25mM each final)
    • 5 μL 10X reaction buffer
    • 1.56 μL 3.2 nM ΦX174 template (0.1 nM final)
    • 1 μL PfuUltra I DNA polymerase
  • Aliquot 2 x 18 μL, and add:
    • 2 μL 100 μM primer 4 mix
    • 2 μL 10 μM primer 4 mix
  • Cycling parameters:
    1. 95 °C 2 min
    2. 95 °C 30 s
    3. 58° C 30 s
    4. 68 °C 12 min
    5. Repeat 2-4 an additional 29 times for 30 total cycles
    6. 72 °C 30 min
    7. 12 °C hold