User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/09/18

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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:
    1. 95 °C 2 min
    2. 95 °C 30 s
    3. 58° C 30 s
    4. 72 °C 12 min
    5. Repeat 2-4 an additional 29 times for 30 total cycles
    6. 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.