User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/09/24

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==Entry title==
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===Hypothesis 2: Gene L is necessary for phage propagation.===
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* Insert content here...
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 +
* Gel electrophoresis results:
 +
** 1 μM primer mix = ~5 kb nicked DNA corresponding to ΦX174 genome
 +
** 10 μM primer mix = NO AMPLIFICATION\
 +
* Ah ha! Since the 1 μM primer mix was diluted from the 10 μM primer mix, it is the case that overly concentrated primers inhibit PCR.
 +
* To optimize this:
 +
* 50 μL + 10% WP-PCR reaction:
 +
** 37 μL H<sub>2</sub>O
 +
** 5.5 μL 10X reaction buffer
 +
** 1.65 μL 3.2 nM ΦX174 template (~ 0.1 nM final)
 +
** 0.55 μL 25 mM dNTPs mix
 +
** 1.1 μL PfuUltra I DNA polymerase
 +
* Aliquot 5 × 9 μL:
 +
*# 1 μL water = 0 μM primer 4
 +
*# 1 μL 12.5 μM primer mix = 1.25 μM primer 4
 +
*# 1 μL 25 μM primer mix = 2.5 μM primer 4
 +
*# 1 μL 50 μM primer mix = 5 μM primer 4
 +
*# 1 μL 100 μM primer mix = 100 μM primer 4
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* Cycling parameters:
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*# 95 °C 2 m
 +
*# 95 °C 30 s
 +
*# 58° C 30 s
 +
*# 72 °C 15 m
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*# Repeat 2-4 an additional 29 times for 30 total cycles
 +
*# 72 °C 30 m
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__NOTOC__
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-----------------
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 +
* 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 17:35, 24 September 2012

PHIX174 Cell Free Expression Main project page
Previous entry      Next entry

Hypothesis 2: Gene L is necessary for phage propagation.

  • Gel electrophoresis results:
    • 1 μM primer mix = ~5 kb nicked DNA corresponding to ΦX174 genome
    • 10 μM primer mix = NO AMPLIFICATION\
  • Ah ha! Since the 1 μM primer mix was diluted from the 10 μM primer mix, it is the case that overly concentrated primers inhibit PCR.
  • To optimize this:
  • 50 μL + 10% WP-PCR reaction:
    • 37 μL H2O
    • 5.5 μL 10X reaction buffer
    • 1.65 μL 3.2 nM ΦX174 template (~ 0.1 nM final)
    • 0.55 μL 25 mM dNTPs mix
    • 1.1 μL PfuUltra I DNA polymerase
  • Aliquot 5 × 9 μL:
    1. 1 μL water = 0 μM primer 4
    2. 1 μL 12.5 μM primer mix = 1.25 μM primer 4
    3. 1 μL 25 μM primer mix = 2.5 μM primer 4
    4. 1 μL 50 μM primer mix = 5 μM primer 4
    5. 1 μL 100 μM primer mix = 100 μM primer 4
  • 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
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