User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/08/18

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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 Ta 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 75 s
    5. Repeat 2-4 an additional 29 times = 30 cycles of PCR
    6. 72 °C 3 min
    7. 12 °C hold