Difference between revisions of "User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/09/23"

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m (Hypothesis 2: Gene L is necessary for phage propagation.)
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*# 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 m
 
*# 72 °C 30 m
 
-----------------
 
 
* 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 14:21, 24 September 2012

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Hypothesis 2: Gene L is necessary for phage propagation.

  • Made new primer 4 and primer 4 T3485A mixes from the source at 100 and 10 μM.
  • 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