User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/09/23

Hypothesis 2: Gene L is necessary for phage propagation.

• Made new primer 4 and primer 4 T3485A mixes from the source at 100 and 10 μM.
• 50 μL WP-PCR reaction:
  • 37 μL H₂O
  • 5 μL 10X reaction buffer
  • 1.5 μL 3.2 nM ΦX174 template (~ 0.1 nM final)
  • 0.5 μL 25 mM dNTPs mix
  • 1 μL PfuUltra I DNA polymerase
• Aliquot 2 × 18 μL:
  1. 2 μL 10 μM primer 4 mix
  2. 2 μL 100 μM primer 4 mix

• Cycling parameters:
  1. 95 °C 2 m
  2. 95 °C 30 s
  3. 58° C 30 s
  4. 72 °C 15 m
  5. Repeat 2-4 an additional 29 times for 30 total cycles
  6. 72 °C 30 m