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

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, T_a, 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: 625/655 nM @ gain = 61 w/ settings
  4. Read: 625/655 nM @ gain = 211 w/ settings
  5. Shake: fast double orbital 30s
  6. Loop: 3-5 every 3 m for 12 h
  • settings = high lamp energy, endpoint, bottom reading, other settings default