User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/08/19

Hypothesis 2: Gene L is necessary for phage propagation.

• Following yesterday's notes, I repeated the whole plasmid PCR protocol for primers 2 (63-mer) and 3 (77-mer) (separate PCRs, since T_a is 63 °C and 63 °C, respectively).

• 60 μL + 10% whole plasmid PCR reaction to test the 63-mer primer pair 2 and the 77-mer primer pair 3.
  1. 45.87 μL H₂O
  2. 6.6 μL 10X reaction buffer
  3. 8.25 μL 2mM dNTPs (each) (0.25mM each final)
  4. 1.32 μL template (0.1nM final)
  5. 2.64 μL 5 μM each forward/reverse primer mix (200nM each final)
  6. 1.32 μL PfuUltra II fusion HS DNA polymerase
• I divided the reaction 4×14.1 μL and then made the following 15 μL reactions
  1. -template, primer 2 (0.3 μL water, 0.6 μL primer 1 mix of 5 μM each S and AS primer 2)
  2. +template, primer 2 (0.3 μL water, 0.6 μL primer 1 mix of 5 μM each S and AS primer 2)
  3. -template, primer 3 (0.3 μL water, 0.6 μL primer 4 mix of 5 μM each S and AS primer 3)
  4. +template, primer 3 (0.3 μL water, 0.6 μL primer 4 mix of 5 μM each S and AS primer 3)
• I then performed whole plasmid PCR with the following cycling parameters:
  1. 95 °C 2 min
  2. 95 °C 20 s
  3. 63 °C 20 s (2) or 66 °C 20 s (3)
  4. 72 °C 15 s
  5. Repeat 2-4 an additional 29 times = 30 cycles of PCR
  6. 72 °C 3 min
  7. 12 °C hold