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

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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.
    1. 0 nM - 2.82 ng/μL = 0.82 nM = 0.85 fold amplification
    2. 100 nM - 245 ng/μL = 71 nM = 730 fold amplification
    3. 200 nM - 270 ng/μL = 78 nM = 810 fold amplification
    4. 500 nM - 303 ng/μL = 88 nM = 910 fold amplification
    5. 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.
    1. water = 0 nM
    2. 1 μL 10μM primer 4 mix (each) = 100 nM
    3. 1 μL 20μM primer 4 mix (each) = 200 nM
    4. 1 μL 50μM primer 4 mix (each) = 500 nM
    5. 1 μL 100μM primer 4 mix (each) = 1000 nM
  • Cycling parameters:
    1. 95 °C 2 min
    2. 95 °C 20 s
    3. 58 °C 20 s
    4. 72 °C 90 s
    5. Repeat 2-4 an additional 29 times = 30 cycles
    6. 72 °C 3 min
    7. 12 °C hold