User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/09/21

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Hypothesis 2: Gene L is necessary for phage propagation.

  • Unfortunately, gel electrophoresis indicated both primer 4 and primer 4 T3485A samples did not work.
  • Here is a list of things that changed from yesterday until today:
    • new dNTPs mix: shouldn't be a problem, so will continue to use this mix.
    • re-programmed PCR program: I checked the machine. The program is as I have it here, so it should work, since the same (or similar) cycling parameters worked previously.
    • different primer mixes: this I'm not so sure of. I will create new mixes at 100 μM and 10 μM and try again.
  • 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:
    1. 2 μL 10 μM primer 4 mix
    2. 2 μL 100 μM primer 4 mix
  • 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, 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


Characterization B-C: Expression of PHIX174 promoters/UTRs fused to PX-UTR1-deGFP and PX-UTRX-deGFP.

  • Gel electrophoresis indicated BamXI/XhoI double digested DNA of:
    1. pBEST-PA-MGapt-T500 ~ 2500 bp dsDNA - OK
    2. pBEST-PB-MGapt-T500 ~ 2500 bp dsDNA - OK
    3. pBEST-PD-MGapt-T500 ~ 2500 bp dsDNA - OK
    4. pBEST-PF-MGapt-T500 ~ 3000-3500 bp dsDNA - not OK
    5. pBEST-PG-MGapt-T500 ~ 2500 bp dsDNA - OK
    6. pBEST-PL-MGapt-T500 ~ 2500 bp dsDNA - OK
    7. pBEST-PL-PA-MGapt-T500 ~ smear - not OK
    8. pBEST-NONE-MGapt-T500 ~ 2500 bp dsDNA - OK
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