Hypothesis 2: Gene L is necessary for phage propagation.
- Gel results from WP-PCR on 09/17/2012 showed amplification (at ~> 5 kb dsDNA ladder, which is correct)! So, either it was the 68 °C extension temperature, a bad tube of primer mix, or both.
- Next step is to test parallel PFU ligase reaction to repair nicked DNA.
- 50 μL WP-PCR reaction:
- 30.2 μL H2O
- 6.25 μL 2mM dNTPs (each) (0.25mM each final)
- 5 μL 10X reaction buffer
- 5 μL 20 μM primer 4 mix
- 1.56 μL 3.2 nM ΦX174 template (0.1 nM final)
- 1 μL PfuUltra I DNA polymerase
- Aliquot 2 × 19.6 μL
- +ligase: 0.4 μL PFU ligase
- -ligase: 0.4 μL water
- Cycling parameters:
- 95 °C 2 m
- 95 °C 30 s
- 58° C 30 s
- 72 °C 12 m
- 55 °C 1 m
- Repeat 2-4 an additional 29 times for 30 total cycles
- 72 °C 30 m
- 55 °C 1 h
Characterization B-C: Expression of PHIX174 promoters/UTRs fused to PX-UTR1-deGFP and PX-UTRX-deGFP.
- 50 mL midiculure of KL740 harboring pBEST-Pr-MGapt-UTR1-deGFP-T500 at 29 °C.
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PHIX174 Cell Free Expression
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