User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/08/19

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Hypothesis 2: Gene L is necessary for phage propagation.

  • Here is the quantiluore results from the WP-PCR carried over the past two days with the differently lengthed primers. ΦX174 5 nM was 10.9 ng/μL = 3.2 nM actual. In the reactions, this meant the template was 3.2 nM/50 = 0.063 nM. So concentration of template is low by about 40% in previous experiments. This will be corrected in the future so that 0.1 nM actual template will be used.
    1. 45-mer with Ta = 58 °C: -template = 8.3 ng/μL = 577 nM (using primer species for conversion), +template (using ΦX174 for conversion) = 42.7 ng/μL = 12.4 nM = 565 fold amplification.
    2. 63-mer with Ta = 63 °C: -template = 7.2 ng/μL = 342 nM, +template = 7.3 ng/μL = 2.5 nM = 33 fold amplification.
    3. 77-mer with Ta = 66 °C: -template = 36.5 ng/μL = 1481 nM , +template = 17.5 ng/μL = 5.1 nM = 80 fold amplification. It looks as if the -template and +template samples could have been switched. If this was the case, then the results change to -template = 17.5 ng/μL = 709 nM, +template = 36.5 ng/μL = 10.6 nM = 167 fold amplification.
    4. 36-mer with Ta = 58 °C: -template = 9.6 ng/μL = 908 nM , +template = 123.6 ng/μL = 35.9 nM = 565 fold amplification.
  • The 36-mer (primer 4) showed the best yield, so I ordered the mutagenic version of this primer from BMGC.
    • ΦX174 4 S T3845A: catggtattgataaagctgtAgccgatacttggaac
    • ΦX174 4 AS T3845A: gttccaagtatcggcTacagctttatcaataccatg
  • Next step is to optimize primer concentration over a range of 0, 100, 200, 500, 1000, and 2000 nM (each). I made 10X primer 4 mixes over this concentration range (each). 60 μL + 10% WP-PCR reaction.
    • 41.4 μL H2O
    • 6.6 μL 10X reaction buffer
    • 8.25 μL 2mM dNTPs (each) (0.25mM each final)
    • 1.875 μL 3.2 nM ΦX174 template (0.1nM final)
    • 1.32 μL PfuUltra II fusion HS DNA polymerase
  • Aliquot 6×9 μL.
    1. water = 0 nM
    2. 1 μL 1μM primer 4 mix (each) = 100 nM
    3. 1 μL 2μM primer 4 mix (each) = 200 nM
    4. 1 μL 5μM primer 4 mix (each) = 500 nM
    5. 1 μL 10μM primer 4 mix (each) = 1000 nM
    6. 1 μL 20μM primer 4 mix (each) = 2000 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
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