User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/09/17

PHIX174 Cell Free Expression

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Hypothesis 2: Gene L is necessary for phage propagation. Gel results from WP-PCR on 09/14/2012 showed no amplification. I have a new enzyme now, PfuUltra1, so I will do a simple test to see if I can get amplification in the first place. 50 μL WP-PCR reaction: 31.2 μL H2O 6.25 μL 2mM dNTPs (each) (0.25mM each final) 5 μL 10X reaction buffer 1.56 μL 3.2 nM ΦX174 template (0.1 nM final) 1 μL PfuUltra I DNA polymerase Aliquot 2 x 18 μL, and add: 2 μL 100 μM primer 4 mix 2 μL 10 μM primer 4 mix Cycling parameters: 95 °C 2 min 95 °C 30 s 58° C 30 s 68 °C 12 min Repeat 2-4 an additional 29 times for 30 total cycles 72 °C 30 min 12 °C hold