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

Hypothesis 2: Gene L is necessary for phage propagation.

• Last time, I verified the final WP-PCR protocol.
• Now, I want to see if DpnI digestion works or not on the methylated ΦX174 template genomic DNA in the PCR conditions.
• 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.
• 50 μL WP-PCR reaction w/o primers:
  • 37 μL H₂O
  • 7.5 μL 100 nM ΦX174 template (~15 nM final)
  • 5 μL 10X reaction buffer
  • 0.5 μL 25 mM dNTPs mix
  • 1 μL PfuUltra I DNA polymerase
• 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
• I prepared this today (Fri) and placed in the Chromato. Vincent will run the WP-PCR Mon. morning.

• Next on the list:
  1. 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.

• 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