User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/09/21

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m (Hypothesis 2: Gene L is necessary for phage propagation.)
Current revision (21:38, 23 September 2012) (view source)
m (Hypothesis 2: Gene L is necessary for phage propagation.)
 
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** re-programmed PCR program: I checked the machine. The program is as I have it here, so it should work, since the same (or similar) cycling parameters worked previously.
** re-programmed PCR program: I checked the machine. The program is as I have it here, so it should work, since the same (or similar) cycling parameters worked previously.
** different primer mixes: this I'm not so sure of. I will create new mixes at 100 μM and 10 μM and try again.
** different primer mixes: this I'm not so sure of. I will create new mixes at 100 μM and 10 μM and try again.
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* 50 μL WP-PCR reaction:
 
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** 37 μL H<sub>2</sub>O
 
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** 5 μL 10X reaction buffer
 
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** 1.5 μL 3.2 nM ΦX174 template (~ 0.1 nM final)
 
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** 0.5 μL 25 mM dNTPs mix
 
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** 1 μL PfuUltra I DNA polymerase
 
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* Aliquot 2 × 18 μL:
 
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*# 2 μL 10 μM primer 4 mix
 
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*# 2 μL 100 μM primer 4 mix
 
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* Cycling parameters:
 
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*# 95 °C 2 m
 
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*# 95 °C 30 s
 
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*# 58° C 30 s
 
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*# 72 °C 15 m
 
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*# Repeat 2-4 an additional 29 times for 30 total cycles
 
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*# 72 °C 30 m
 
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-----------------
 
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* Next on the list:
 
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*# DpnI digestion (w/ and w/o PCR purification)
 
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*# Topisomerase IV / Gyrase (preceded by PCR purification) - how to assay linking number?
 
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* 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
 
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** experimental 1 = +template, +primer 4, +DNAP
 
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** experimental 2 = +template. +primer 4 T3585A, +DNAP
 
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** control 1: -template, +primer 4, +DNAP
 
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** control 2: -template, +primer 4 T3485, +DNAP
 
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** control 3: +template, +primer 4, +DNAP
 
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** control 4: +template, +primer 4 T3485, +DNAP
 
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** control 5: +template, -primers, +DNAP
 
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* Followed by:
 
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** (possible purification and then) DpnI digestion
 
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** PCR purification
 
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** Linking number adjustment by gyrase / topisomerase IV
 
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** PCR purification
 
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===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.===

Current revision

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

  • Unfortunately, gel electrophoresis indicated both primer 4 and primer 4 T3485A samples did not work.
  • Here is a list of things that changed from yesterday until today:
    • new dNTPs mix: shouldn't be a problem, so will continue to use this mix.
    • re-programmed PCR program: I checked the machine. The program is as I have it here, so it should work, since the same (or similar) cycling parameters worked previously.
    • different primer mixes: this I'm not so sure of. I will create new mixes at 100 μM and 10 μM and try again.

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

  • Gel electrophoresis indicated BamXI/XhoI double digested DNA of:
    1. pBEST-PA-MGapt-T500 ~ 2500 bp dsDNA - OK
    2. pBEST-PB-MGapt-T500 ~ 2500 bp dsDNA - OK
    3. pBEST-PD-MGapt-T500 ~ 2500 bp dsDNA - OK
    4. pBEST-PF-MGapt-T500 ~ 3000-3500 bp dsDNA - not OK
    5. pBEST-PG-MGapt-T500 ~ 2500 bp dsDNA - OK
    6. pBEST-PL-MGapt-T500 ~ 2500 bp dsDNA - OK
    7. pBEST-PL-PA-MGapt-T500 ~ smear - not OK
    8. pBEST-NONE-MGapt-T500 ~ 2500 bp dsDNA - OK
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