User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/09/28

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Current revision (16:08, 28 September 2012) (view source)
m (Characterization B-C: Expression of PHIX174 promoters/UTRs fused to PX-UTR1-deGFP and PX-UTRX-deGFP.)
 
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===Hypothesis 2: Gene L is necessary for phage propagation.===
 +
 +
* Last time, I verified the final WP-PCR protocol.
 +
* Now, I want to see if DpnI digestion works or not on the methylated ΦX174 template genomic DNA in the PCR conditions.
 +
* For there to be 0.5 μg of DNA to digest in 10 μL, which is enough to see on a gel, I'll need 50 ng/μL ~ 15 nM final concentration DNA.
 +
* 50 μL WP-PCR reaction w/o primers:
 +
** 36 μL H<sub>2</sub>O
 +
** 7.5 μL 100 nM ΦX174 template (~15 nM final)
 +
** 5 μL 10X reaction buffer
 +
** 0.5 μL 25 mM dNTPs mix
 +
** 1 μL PfuUltra I DNA polymerase
 +
* WP-PCR Cycling parameters:
 +
*# 95 °C 2 m
 +
*# 95 °C 30 s
 +
*# 58° C 30 s
 +
*# 72 °C 15 m
 +
*# Repeat 2-4 an additional 29 times for 30 total cycles
 +
*# 72 °C 30 m
 +
* I prepared this today (Fri) and placed in the Chromato. Vincent will run the WP-PCR Mon. morning.
 +
* Used up the rest of the PfuUltra I DNAP making 225 μL 90% ΦX174 WP-PCR mix
 +
** 185 μL H<sub>2</sub>O
 +
** 25 μL 10X reaction buffer
 +
** 7.5 μL 3.2 nM ΦX174 template (~0.1 nM final)
 +
** 2.5 μL 25 mM dNTPs mix
 +
** 5 μL PfuUltra I DNA polymerase
 +
 +
-----------------
 +
 +
* Next on the list:
 +
*# 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
 +
===Characterization B-C: Expression of PHIX174 promoters/UTRs fused to PX-UTR1-deGFP and PX-UTRX-deGFP.===
===Characterization B-C: Expression of PHIX174 promoters/UTRs fused to PX-UTR1-deGFP and PX-UTRX-deGFP.===
* Experiment specified yesterday was actually run today, with the modification that reads happen every minute for 12 h, with continuous fast double-orbital shaking between read times.
* Experiment specified yesterday was actually run today, with the modification that reads happen every minute for 12 h, with continuous fast double-orbital shaking between read times.

Current revision

PHIX174 Cell Free Expression Main project page
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Hypothesis 2: Gene L is necessary for phage propagation.

  • Last time, I verified the final WP-PCR protocol.
  • Now, I want to see if DpnI digestion works or not on the methylated ΦX174 template genomic DNA in the PCR conditions.
  • For there to be 0.5 μg of DNA to digest in 10 μL, which is enough to see on a gel, I'll need 50 ng/μL ~ 15 nM final concentration DNA.
  • 50 μL WP-PCR reaction w/o primers:
    • 36 μL H2O
    • 7.5 μL 100 nM ΦX174 template (~15 nM final)
    • 5 μL 10X reaction buffer
    • 0.5 μL 25 mM dNTPs mix
    • 1 μL PfuUltra I DNA polymerase
  • WP-PCR 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
  • I prepared this today (Fri) and placed in the Chromato. Vincent will run the WP-PCR Mon. morning.
  • Used up the rest of the PfuUltra I DNAP making 225 μL 90% ΦX174 WP-PCR mix
    • 185 μL H2O
    • 25 μL 10X reaction buffer
    • 7.5 μL 3.2 nM ΦX174 template (~0.1 nM final)
    • 2.5 μL 25 mM dNTPs mix
    • 5 μL PfuUltra I DNA polymerase

  • Next on the list:
    1. 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


Characterization B-C: Expression of PHIX174 promoters/UTRs fused to PX-UTR1-deGFP and PX-UTRX-deGFP.

  • Experiment specified yesterday was actually run today, with the modification that reads happen every minute for 12 h, with continuous fast double-orbital shaking between read times.
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