- Next on the list:
- DpnI digestion (w/ and w/o PCR purification)
- 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:
- 2 μL water = 0 nM
- 2 μL 50 nM / 3^4 * 20% = .123 nM
- 2 μL 50 nM / 3^3 * 20% = .370 nM
- 2 μL 50 nM / 3^2 * 20% = 1.11 nM
- 2 μL 50 nM / 3^1 * 20% = 3.33 nM
- 2 μL 45.7 nM / 3^0 * 20% = 9.14 nM
- Plate reader:
- 29 °C
- Shake: fast double orbital 30s
- Read: 625/655 nM @ gain = 175 w/ settings
- Read: 625/655 nM @ gain = 200 w/ settings
- Read: 625/655 nM @ gain = 225 w/ settings
- Loop: 2-5 every 3 m for 12 h
- settings = full plate, high lamp energy, endpoint, bottom reading, other settings default
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PHIX174 Cell Free Expression
