User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/09/14

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Hypothesis 2: Gene L is necessary for phage propagation.

  • 50 μL WP-PCR reaction:
    • 30.2 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 DNAP
  • Aliquot 2 × 19.6 μL, and add:
    • 0.4 μL water (to be 1-2)
    • 0.4 μL Pfu Ligase (to be 3-4)
  • Aliquot 2 × 2 × 9 μL, and add:
    • 1 μL 100 μM primer 4 mix (each) (1-2)
    • 1 μL 100 μM primer 4 T3485A mix (each) (3-4)
  • Cycling parameters:
    1. 95 °C 2 min
    2. 95 °C 20 s
    3. 58° C 20 s
    4. 72 °C 12 min
    5. 55 °C 1 min
    6. Repeat 2-4 an additional 29 times for 30 total cycles
    7. 72 °C 30 min
    8. 55 °C 60 min
    9. 12 °C hold
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