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

PHIX174 Cell Free Expression

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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: 2 μL 10 μM primer 4 mix 2 μL 100 μM primer 4 mix Cycling parameters: 95 °C 2 m 95 °C 30 s 58° C 30 s 72 °C 15 m Repeat 2-4 an additional 29 times for 30 total cycles 72 °C 30 m