User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/09/25: Difference between revisions
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== | ===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 = nM. | |||
* 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) | |||
** 12.1 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) | |||
** 4.5 μL PEG8000 40% (2% final) | |||
* Aliquot 6 × 9 μL into 394-well plate and then add plasmid (pBEST-Pr-MGapt-UTR1-deGFP-T500) range: | |||
*# 1 μL water = 0 nM | |||
*# 1 μL 50 nM / 3^4 * 20% = .123 nM | |||
*# 1 μL 50 nM / 3^3 * 20% = .370 nM | |||
*# 1 μL 50 nM / 3^2 * 20% = 1.11 nM | |||
*# 1 μL 50 nM / 3^1 * 20% = 3.33 nM | |||
*# 1 μL 50 nM / 3^0 * 20% = 10 nM | |||
* Plate reader: |
Revision as of 11:10, 25 September 2012
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Characterization B-C: Expression of PHIX174 promoters/UTRs fused to PX-UTR1-deGFP and PX-UTRX-deGFP.
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