User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/08/20

PHIX174 Cell Free Expression

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Hypothesis 2: Gene L is necessary for phage propagation. Here is the quantiluore results from the WP-PCR of 0.1 nM ΦX174 with variable 36-mer primer concentration. ΦX174 1 nM template only control gave 3.34 ng/μL = 0.97 nM. WAIT: Shouldn't this be 0.1 nM? It seems like I am measuring things to be one order of magnitude too high. 0 nM - 2.82 ng/μL = 0.82 nM = 0.85 fold amplification 100 nM - 245 ng/μL = 71 nM = 730 fold amplification 200 nM - 270 ng/μL = 78 nM = 810 fold amplification 500 nM - 303 ng/μL = 88 nM = 910 fold amplification 1000 nM - 320 ng/μL = 93 nM = 960 fold amplification Next step is to optimize primer concentration over a range of 0, 1, 2, 5, and 10 μM (each). I made 10X primer 4 mixes (0, 10, 20, 50, and 100 μM) over this concentration range. 50 μL + 10% WP-PCR reaction. 39.6 μL H2O 5.5 μL 10X reaction buffer 6.88 μL 2mM dNTPs (each) (0.25mM each final) 1.875 μL 3.2 nM ΦX174 template (0.1nM final) 1.1 μL PfuUltra II fusion HS DNA polymerase Aliquot 5×9 μL. water = 0 nM 1 μL 10μM primer 4 mix (each) = 100 nM 1 μL 20μM primer 4 mix (each) = 200 nM 1 μL 50μM primer 4 mix (each) = 500 nM 1 μL 100μM primer 4 mix (each) = 1000 nM Cycling parameters: 95 °C 2 min 95 °C 20 s 58 °C 20 s 72 °C 90 s Repeat 2-4 an additional 29 times = 30 cycles 72 °C 3 min 12 °C hold