User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/09/24

Hypothesis 2: Gene L is necessary for phage propagation.

• Gel electrophoresis results:
  • 1 μM primer mix = ~5 kb nicked DNA corresponding to ΦX174 genome
  • 10 μM primer mix = NO AMPLIFICATION\
• Ah ha! Since the 1 μM primer mix was diluted from the 10 μM primer mix, it is the case that overly concentrated primers inhibit PCR.
• To optimize this:
• 50 μL + 10% WP-PCR reaction:
  • 37 μL H₂O
  • 5.5 μL 10X reaction buffer
  • 1.65 μL 3.2 nM ΦX174 template (~ 0.1 nM final)
  • 0.55 μL 25 mM dNTPs mix
  • 1.1 μL PfuUltra I DNA polymerase
• Aliquot 5 × 9 μL:
  1. 1 μL water = 0 μM primer 4
  2. 1 μL 12.5 μM primer mix = 1.25 μM primer 4
  3. 1 μL 25 μM primer mix = 2.5 μM primer 4
  4. 1 μL 50 μM primer mix = 5 μM primer 4
  5. 1 μL 100 μM primer mix = 100 μM primer 4

• Cycling parameters:
  1. 95 °C 2 m
  2. 95 °C 30 s
  3. 58° C 30 s
  4. 72 °C 15 m
  5. Repeat 2-4 an additional 29 times for 30 total cycles
  6. 72 °C 30 m

• Next on the list:
  1. DpnI digestion (w/ and w/o PCR purification)
  2. 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, T_a, 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