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.=== | ===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. | * 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. | * 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%: | * Cell-free expression 80%: | ||
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*# 2 μL 50 nM / 3^2 * 20% = 1.11 nM | *# 2 μL 50 nM / 3^2 * 20% = 1.11 nM | ||
*# 2 μL 50 nM / 3^1 * 20% = 3.33 nM | *# 2 μL 50 nM / 3^1 * 20% = 3.33 nM | ||
*# 2 μL | *# 2 μL 45.7 nM / 3^0 * 20% = 9.14 nM | ||
* Plate reader: | * Plate reader: | ||
*# 29 °C | *# 29 °C |
Revision as of 14:59, 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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