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

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 = full plate, high lamp energy, endpoint, bottom reading, other settings default