User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/09/20: Difference between revisions

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===Hypothesis 2: Gene L is necessary for phage propagation.===
* Unfortunately, both + and - PFU ligase samples showed amplification at ~> 5.3 kb dsDNA, so the ligase was not able to repair the nick to the amplified PHIX174 genome in parallel in the reaction.
* Next step is to make amplify a large batch of PHIX174 and PHIX174 T3485A.
* 2 × 50 μL WP-PCR reaction:
** 37 μL H<sub>2</sub>O
** 5 μL 10X reaction buffer
** 5 μL 100 μM primer 4 mix OR 20 μM primer 4 T3485A mix
** 2 μL 3.2 nM ΦX174 template (~ 0.1 nM final)
** 1 μL PfuUltra I DNA polymerase
* Cycling parameters:
*# 95 °C 2 m
*# 95 °C 30 s
*# 58° C 30 s
*# 72 °C 20 m
*# Repeat 2-4 an additional 29 times for 30 total cycles
*# 72 °C 40 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
===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.===


Revision as of 13:39, 20 September 2012

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Hypothesis 2: Gene L is necessary for phage propagation.

  • Unfortunately, both + and - PFU ligase samples showed amplification at ~> 5.3 kb dsDNA, so the ligase was not able to repair the nick to the amplified PHIX174 genome in parallel in the reaction.
  • Next step is to make amplify a large batch of PHIX174 and PHIX174 T3485A.
  • 2 × 50 μL WP-PCR reaction:
    • 37 μL H2O
    • 5 μL 10X reaction buffer
    • 5 μL 100 μM primer 4 mix OR 20 μM primer 4 T3485A mix
    • 2 μL 3.2 nM ΦX174 template (~ 0.1 nM final)
    • 1 μL PfuUltra I DNA polymerase
  • Cycling parameters:
    1. 95 °C 2 m
    2. 95 °C 30 s
    3. 58° C 30 s
    4. 72 °C 20 m
    5. Repeat 2-4 an additional 29 times for 30 total cycles
    6. 72 °C 40 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

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

  • Spun down last night's midiculture in ~10 2 mL tubes (4 mL/tube) and stored at -40 °C for later use.
  • Verification of pBEST-PX-MGapt plasmid set. I have 8 plasmids (6 promoters, 1 PL-PA tandem promoter, and 1 promoterless control). Verify by BamHI-XhoI double digest across the board.
  • 100 μL BamHI-XhoI double digest @ 90%:
    • 60 μL H2O
    • 10 μL NEB Buffer 3
    • 10 μL 10 mg/mL BSA
    • 5 μL BamHI
    • 5 μL XhoI
  • Aliquot 8 × 9 μL
    1. 1 μL pBEST-PA-MGapt-T500
    2. 1 μL pBEST-PB-MGapt-T500
    3. 1 μL pBEST-PD-MGapt-T500
    4. 1 μL pBEST-PF-MGapt-T500
    5. 1 μL pBEST-PG-MGapt-T500
    6. 1 μL pBEST-PL-MGapt-T500
    7. 1 μL pBEST-PL-PA-MGapt-T500
    8. 1 μL pBEST-NONE-MGapt-T500
  • Incubate 37 °C overnight.
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