Hypothesis 2: Gene L is necessary for phage propagation.
- Gel results from WP-PCR on 09/17/2012 showed no amplification. Discussed with Vincent. We will go over my protocols and results in the lab meeting.
- I checked the PCR program and noticed I used 68 °C for elongation, instead of 72 °C like I thought.
- One more try. This time I will use 10 μM primer mix, which worked previously.
- 25 μL WP-PCR reaction:
- 15.6 μL H2O
- 3.12 μL 2mM dNTPs (each) (0.25mM each final)
- 2.5 μL 10X reaction buffer
- 2.5 μL 10 μM primer 4 mix
- 0.78 μL 3.2 nM ΦX174 template (0.1 nM final)
- 0.5 μL PfuUltra I DNA polymerase
- Cycling parameters:
- 95 °C 2 min
- 95 °C 30 s
- 58° C 30 s
- 72 °C 12 min
- Repeat 2-4 an additional 29 times for 30 total cycles
- 72 °C 30 min
Characterization B-C: Expression of PHIX174 promoters/UTRs fused to PX-UTR1-deGFP and PX-UTRX-deGFP.
- Many problems experienced over the summer regarding construction of PX-UTR1-deGFP and PX-UTRX-deGFP.
- Transformed pBEST-Pr-MGapt-UTR1-deGFP-T500 into KL740.