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

PHIX174 Cell Free Expression

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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. 45.87 μL H2O 6.6 μL 10X reaction buffer 8.25 μL 2mM dNTPs (each) (0.25mM each final) 1.32 μL template (0.1nM final) 2.64 μL 5 μM each forward/reverse primer mix (200nM each final) 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 -template, primer 2 (0.3 μL water, 0.6 μL primer 1 mix of 5 μM each S and AS primer 2) +template, primer 2 (0.3 μL water, 0.6 μL primer 1 mix of 5 μM each S and AS primer 2) -template, primer 3 (0.3 μL water, 0.6 μL primer 4 mix of 5 μM each S and AS primer 3) +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: 95 °C 2 min 95 °C 20 s 63 °C 20 s (2) or 66 °C 20 s (3) 72 °C 75 s Repeat 2-4 an additional 29 times = 30 cycles of PCR 72 °C 3 min 12 °C hold