User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/09/27

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m (Hypothesis 2: Gene L is necessary for phage propagation.)
Current revision (16:07, 28 September 2012) (view source)
m (Hypothesis 2: Gene L is necessary for phage propagation.)
 
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===Hypothesis 2: Gene L is necessary for phage propagation.===
 
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* Last time, I verified the final WP-PCR protocol.
 
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* Now, I want to see if DpnI digestion works or not on the methylated ΦX174 template genomic DNA in the PCR conditions.
 
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* 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.
 
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* 50 μL WP-PCR reaction w/o primers:
 
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** 36 μL H<sub>2</sub>O
 
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** 7.5 μL 100 nM ΦX174 template (~15 nM final)
 
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** 5 μL 10X reaction buffer
 
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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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* WP-PCR 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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* I prepared this today (Fri) and placed in the Chromato. Vincent will run the WP-PCR Mon. morning.
 
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* Used up the rest of the PfuUltra I DNAP making 225 μL 90% ΦX174 WP-PCR mix
 
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** 185 μL H<sub>2</sub>O
 
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** 25 μL 10X reaction buffer
 
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** 7.5 μL 3.2 nM ΦX174 template (~0.1 nM final)
 
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** 2.5 μL 25 mM dNTPs mix
 
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** 5 μL PfuUltra I DNA polymerase
 
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* Next on the list:
 
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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
 
===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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Characterization B-C: Expression of PHIX174 promoters/UTRs fused to PX-UTR1-deGFP and PX-UTRX-deGFP.

  • Graphs of MGapt and deGFP vs. time from yesterday's experiment are simply beautiful.
  • However, the data is very noisy at the 1.11 nM level or below, even though the deGFP graphs show that expression is occurring down to at least the .37 nM level.
  • Therefore, I will do a range of malachite green at 1 nM, to try and improve the signal at lower plasmid concentrations.
  • 80 % cell free expression reaction
    • 30 μL extract (12May11)
    • 1.17 μL water
    • 0.9 μL 100 mM Mg-glutamate (1 mM final)
    • 2 μL 3 M K-glutamate (66.7 mM final)
    • 22.5 μL 6 mM amino acids mix (1.5 mM final)
    • 6.43 μL 14X 3-PGC buffer (1X final)
    • 4.5 μL PEG8000 40% (2% final)
    • 4.5 μL 20 nM plasmid (1 nM final), plasmid = pBEST-Pr-MGapt-UTR1-deGFP-T500
  • Aliquot 6 × 8 μL into 394-well plate and then add malachite green range:
    1. 2 μL water = 0 μM
    2. 2 μL 500 μM / 3^4 * 20% = 0.410 μM
    3. 2 μL 500 μM / 3^4 * 20% = 1.23 μM
    4. 2 μL 500 μM / 3^3 * 20% = 3.70 μM
    5. 2 μL 500 μM / 3^2 * 20% = 11.1 μM
    6. 2 μL 500 μM / 3^1 * 20% = 33.3 μM
    7. 2 μL 500 μM / 3^0 * 20% = 100 μM
  • Plate reader:
    1. Shake: fast double orbital 30s
    2. Read MGapt: 625/655 nM @ gain = 150 w/ settings
    3. Read deGFP: 485/528 nM @ gain = 61 w/ settings
    4. Shake: fast double orbital 30s
    5. Loop: 2-4 every 3 m for 12 h
    • settings = high lamp energy, endpoint, bottom reading, other settings default
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