Hypothesis 2: Gene L is necessary for phage propagation.
- Made new primer 4 and primer 4 T3485A mixes from the source at 100 and 10 μM.
- 50 μL WP-PCR reaction:
- 37 μL H2O
- 5 μL 10X reaction buffer
- 1.5 μL 3.2 nM ΦX174 template (~ 0.1 nM final)
- 0.5 μL 25 mM dNTPs mix
- 1 μL PfuUltra I DNA polymerase
- Aliquot 2 × 18 μL:
- 2 μL 10 μM primer 4 mix
- 2 μL 100 μM primer 4 mix
- Cycling parameters:
- 95 °C 2 m
- 95 °C 30 s
- 58° C 30 s
- 72 °C 15 m
- Repeat 2-4 an additional 29 times for 30 total cycles
- 72 °C 30 m
- Next on the list:
- DpnI digestion (w/ and w/o PCR purification)
- 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
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