User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/09/19: Difference between revisions

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* Cycling parameters:
* Cycling parameters:
*# 95 °C 2 min
*# 95 °C 2 m
*# 95 °C 30 s
*# 95 °C 30 s
*# 58° C 30 s
*# 58° C 30 s
*# 72 °C 12 min
*# 72 °C 12 m
*# 55 °C 1 min
*# 55 °C 1 m
*# Repeat 2-4 an additional 29 times for 30 total cycles
*# Repeat 2-4 an additional 29 times for 30 total cycles
*# 72 °C 30 min
*# 72 °C 30 m
*# 55 °C 1 hr
*# 55 °C 1 h


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Revision as of 14:16, 19 September 2012

PHIX174 Cell Free Expression <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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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
    1. +ligase: 0.4 μL PFU ligase
    2. -ligase: 0.4 μL water
  • Cycling parameters:
    1. 95 °C 2 m
    2. 95 °C 30 s
    3. 58° C 30 s
    4. 72 °C 12 m
    5. 55 °C 1 m
    6. Repeat 2-4 an additional 29 times for 30 total cycles
    7. 72 °C 30 m
    8. 55 °C 1 h
  • 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.

  • 50 mL midiculure of KL740 harboring pBEST-Pr-MGapt-UTR1-deGFP-T500 at 29 °C.
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