User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/08/21

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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 ~0 nM w/ inconsistent readings (not really a problem).
    1. 0 μM - 0.36 nM inconsistent
    2. 1 μM - 0.31 nM inconsistent
    3. 2 μM - 78 nM
    4. 5 μM - 92 nM
    5. 10 μM - 117 nM
    • 100 μM S primer - 39 μM (0.8%)
    • 100 μM AS primer - 60 μM (18%); this needs to be repeated
  • The last three data points satisfies the linear equation product = 4.99x+67.5 with R2 = 0.9997. The fact that this is growing linear indicates that the primers are the limiting component in WP-PCR (as primers typically are in WP-PCR). Next, I will optimize elongation time.
  • Before this, I measured the stock "1" mM S and AS primers by quanitfluore
    1. "1" mM S primer 4 - 0.108 (18%) mM
    2. "1" mM AS primer 4v - 0.081 (7%) mM
  • Still too low by an order of magnitude ... figured it out. The reason for the large systematic and variable errors is that quantifluore uses an intercolating dye and hence OCLY measures dsDNA. ssDNA, like these primers, cannot be measured by quantifluor. Therefore, I trusted the DNA mass cited by the synthesis company, meaning 1 mM primers was the actual value used in calculating primer dilututions.
  • Optimize elongation time. 50 μL WP-PCR reaction:
    • 29.6 μL H2O
    • 5.5 μL 10X reaction buffer
    • 6.88 μL 2mM dNTPs (each) (0.25mM each final)
    • 5.5 μL 10 μM primer 4 mix (each)
    • 1.562 μL 3.2 nM ΦX174 template (0.1nM final)
    • 1 μL PfuUltra II fusion HS DNA polymerase
  • Aliquot 5×10 μL.
  • Cycling parameters:
    1. 95 °C 2 min
    2. 95 °C 20 s
    3. 58 °C 20 s
    4. 72 °C X min, where X = 10:00, 3:20, 1:07, 0:22, and 0:07 (3-fold "serial dilutions of time")
    5. Repeat 2-4 an additional 29 times = 30 cycles
    6. 12 °C hold
  • Note for scheduling assistance ... using PCR step time + 30% estimated ramp time gives estimated total PCR times of the these PCR reactions to be 7:00, 2:40, 1:15, 0:45, and 0:35 (hr:min), respectively.
  • Things that still need to be optimized:
    1. After primer concentrations have been corrected, vary primer concentration by 0, 1E2, 1E3, 1E4, 1E5, 1E6 nM = 1 mM
    2. Ta = 54, 56, 58, 60, 62 °C
  • N cycles = 0, 10, 20, 25, 30, 35, 40, 50
  • After that, tasks include characterizing effects of:
    1. parallel PFU ligation
    2. DpnI digestion (w/ and w/o PCR purification)
    3. Topisomerase IV / Gyrase (preceded by PCR purification) - how to assay linking number?
  • Final experiment is planned to be WP-PCR of 0.1 nM ΦX174 at optimized conditions (primer concentration, Ta, elongation time, N), + PFU ligase
    • experimental 1 = +template, +primer 4, +DNAP
    • experimental 2 = +template. +primer 4 T3585A, +DNAP
    • control 1: -template, +primer 4, +DNAP
    • control 2: -template, +primer 4 T3485, +DNAP
    • control 3: +template, +primer 4, +DNAP
    • control 4: +template, +primer 4 T3485, +DNAP
    • control 5: +template, -primers, +DNAP
  • Followed by:
    • (possible purification and then) DpnI digestion
    • PCR purification
    • Linking number adjustment by gyrase / topisomerase IV
    • PCR purification