User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/09/17: Difference between revisions

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*# 72 °C 30 min
*# 72 °C 30 min
*# 12 °C hold
*# 12 °C hold
-----------------
* Next on the list:
*# DpnI digestion (w/ and w/o PCR purification)
*# 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, T<sub>a</sub>, 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

Revision as of 15:36, 18 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 09/14/2012 showed no amplification. I have a new enzyme now, PfuUltra1, so I will do a simple test to see if I can get amplification in the first place.
  • 50 μL WP-PCR reaction:
    • 31.2 μL H2O
    • 6.25 μL 2mM dNTPs (each) (0.25mM each final)
    • 5 μL 10X reaction buffer
    • 1.56 μL 3.2 nM ΦX174 template (0.1 nM final)
    • 1 μL PfuUltra I DNA polymerase
  • Aliquot 2 x 18 μL, and add:
    • 2 μL 100 μM primer 4 mix
    • 2 μL 10 μM primer 4 mix
  • Cycling parameters:
    1. 95 °C 2 min
    2. 95 °C 30 s
    3. 58° C 30 s
    4. 68 °C 12 min
    5. Repeat 2-4 an additional 29 times for 30 total cycles
    6. 72 °C 30 min
    7. 12 °C hold
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