User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/09/25

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m (Characterization B-C: Expression of PHIX174 promoters/UTRs fused to PX-UTR1-deGFP and PX-UTRX-deGFP.)
m (Characterization B-C: Expression of PHIX174 promoters/UTRs fused to PX-UTR1-deGFP and PX-UTRX-deGFP.)
Line 78: Line 78:
*# 29 °C
*# 29 °C
*# Shake: fast double orbital 30s
*# Shake: fast double orbital 30s
-
*# Read: 625/655 nM @ gain = 61 w/ settings
+
*# Read MGapt: 625/655 nM @ gain = 211 w/ settings
-
*# Read: 625/655 nM @ gain = 211 w/ settings
+
*# Read deGFP: 485/528 nM @ gain = 61 w/ settings
*# Shake: fast double orbital 30s
*# Shake: fast double orbital 30s
-
*# Loop: 3-5 every 3 m for 12 h
+
*# Loop: 2-4 every 3 m for 12 h
** settings = high lamp energy, endpoint, bottom reading, other settings default
** settings = high lamp energy, endpoint, bottom reading, other settings default
 +
* The goal was to measure transcript levels of MGapt and translated deGFP simultaneously.

Revision as of 18:51, 26 September 2012

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

  • Unfortunately, I hosed the 1.25 μM sample (from Monday).
  • Gel electrophoresis showed the following over the rest of the primer 4 mix range:
    • 2.5 μM: a weak band at ~≥ 5 kb (amplified ΦX174 genome), and two stonger bands at ~100 bp and >100 bp
    • 5 μM: two strong bands at ~100 bp and >100 bp
    • 10 μM: two stong bands at ~100 bp and >100 bp
  • Given these results, here's what I think is happening. At too high primer concentration, the ssDNA primers, being complementary strands of WP-PCR, do not unbind, inhibiting PCR.
  • I will do one more test over a primer 4 mix of 0, 0.5, 1, 2, 4, and 8 μM.
  • Aliquot 6 × 4.5 μL ΦX174 WP-PCR mix (1X reaction buffer, .25 mM dNTPs mix, PfuUltra I DNAP, ~0.1 nM ΦX174 template)
    1. 0.5 μL 5 water
    2. 0.5 μL 5 μM primer 4 mix
    3. 0.5 μL 10 μM primer 4 mix
    4. 0.5 μL 20 μM primer 4 mix
    5. 0.5 μL 40 μM primer 4 mix
    6. 0.5 μL 80 μM primer 4 mix
  • 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



  • 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

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

  • Quantifluore of pBEST-Pr-MG-apt-UTR1-deGFP-T500 = 45.7 nM (10.7%).
  • Do cell-free expression in batch-mode to characterize pBEST-Pr-MG-apt-UTR1-deGFP-T500. Idea is to start at lowish gain (say 50), and then increase gain at ~3-4 hr when expression is peaking until an 80% of max reading (80,000 counts of 100,000 max for the PMT) is achieved. This will then be the fixed gain for MGapt experiments.
  • Cell-free expression 80%:
    • 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 200 μM malachite green (10 μM final)
  • Aliquot 6 × 8 μL into 394-well plate and then add plasmid (pBEST-Pr-MGapt-UTR1-deGFP-T500) range:
    1. 2 μL water = 0 nM
    2. 2 μL 50 nM / 3^4 * 20% = .123 nM
    3. 2 μL 50 nM / 3^3 * 20% = .370 nM
    4. 2 μL 50 nM / 3^2 * 20% = 1.11 nM
    5. 2 μL 50 nM / 3^1 * 20% = 3.33 nM
    6. 2 μL 45.7 nM / 3^0 * 20% = 9.14 nM
  • Plate reader:
    1. 29 °C
    2. Shake: fast double orbital 30s
    3. Read: 625/655 nM @ gain = 175 w/ settings
    4. Read: 625/655 nM @ gain = 200 w/ settings
    5. Read: 625/655 nM @ gain = 225 w/ settings
    6. Loop: 2-5 every 3 m for 12 h
    • settings = high lamp energy, endpoint, bottom reading, other settings default
  • Transcription maximized around 1 hr after the experiment began. I fine-tuned the measurements, zeroing in on the range of gain = 210-212 as the maximum possible gain where counts < 80,000. I found that the 10 nM plasmid sample broke that threshold at gain = 212, but didn't at gain = 211.
  • Therefore, gain = 211 is the maximum possible gain, achieving the best possible fluorescence sensitivity, without breaking the 80,000 count threshold at 10 nM plasmid.
  • I then redid the experiment, with the following changes to the plate reader program:
    1. 29 °C
    2. Shake: fast double orbital 30s
    3. Read MGapt: 625/655 nM @ gain = 211 w/ settings
    4. Read deGFP: 485/528 nM @ gain = 61 w/ settings
    5. Shake: fast double orbital 30s
    6. Loop: 2-4 every 3 m for 12 h
    • settings = high lamp energy, endpoint, bottom reading, other settings default
  • The goal was to measure transcript levels of MGapt and translated deGFP simultaneously.
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