User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/09/14: Difference between revisions

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** 6.25 μL 2mM dNTPs (each) (0.25mM each final)
** 6.25 μL 2mM dNTPs (each) (0.25mM each final)
** 5 μL 10X reaction buffer
** 5 μL 10X reaction buffer
** 5 μL 10X primer
** 1.56 μL 3.2 nM ΦX174 template (0.1 nM final)
** 1.56 μL 3.2 nM ΦX174 template (0.1 nM final)
** 1 μL PfuUltra II fusion HS DNA polymerase
** 1 μL PfuUltra II fusion HS DNA polymerase
** 1 μL Pfu Ligase
* Aliquot 2 × 19.6 μL, and add:
* Aliquot 2 × 19.6 μL, and add:
** 0.4 μL water (to be 1-2)
** 0.4 μL water (to be 1-2)
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* Next on the list:
* Next on the list:
*# parallel PFU ligation
*# DpnI digestion (w/ and w/o PCR purification)
*# DpnI digestion (w/ and w/o PCR purification)
*# Topisomerase IV / Gyrase (preceded by PCR purification) - how to assay linking number?
*# Topisomerase IV / Gyrase (preceded by PCR purification) - how to assay linking number?

Revision as of 16:03, 14 September 2012

PHIX174 Cell Free Expression <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

Hypothesis 2: Gene L is necessary for phage propagation.

  • 50 μL:
    • 30.2 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 II fusion HS DNA polymerase
  • Aliquot 2 × 19.6 μL, and add:
    • 0.4 μL water (to be 1-2)
    • 0.4 μL Pfu Ligase (to be 3-4)
  • Aliquot 2 × 2 × 9 μL, and add:
    • 1 μL 100 μM primer 4 mix (each) (1-2)
    • 1 μL 100 μM primer 4 T3485A mix (each) (3-4)
  • Cycling parameters:
    1. 95 °C 2 min
    2. 95 °C 20 s
    3. 58° C 20 s
    4. 72 °C 12 min
    5. 55 °C 1 min
    6. Repeat 2-4 an additional 29 times for 30 total cycles
    7. 72 °C 30 min
    8. 55 °C 60 min
    9. 12 °C hold
  • 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
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