Daniel:Notebook/PosSequencing/2017-6-29
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emPCR 7-Strand Separation (Started Yesterday)
Protocol (From Yesterday)
- Strand Separation (DNA Denaturation)
- Split each sample (1 pM and 100 pM) into 4 parts-> 25 uL each-> put them in 1.5mL tubes
- Add 200 uL KOH buffer to each sample(pH 12.0, 11.5, 11.0, 10.0); denaturation pH of DNA should be around 11.3
- Spin down sample at 13,000rpm for 1 minute
- Remove all but 40uL buffer
- Repeat KOH steps A-C once more
- Add 200 uL 4X SSC buffer (pH 12.0)
- Spin down sample at 13,000rpm for 1 minute
- Remove all but 10uL buffer
- Resuspend in 80 uL 2X SSC buffer
- Single primer qPCR
- Make the following master mixes
- Aliquot 35 uL master mix to appropriate lanes
- Add 2 uL sample according to plate layout below File:PlateLayout20170629-emPCR7-SinglePrimer.png
- Run the following thermocycler program
- 95C 3 min
- 95C 3 sec
- 45C 30 sec
- 72C 20 sec
- plate read
- goto b x90
- 72C 2 min
- 16C hold
Reagent | uL in Single Rxn | Master Mix P2 (12.2X) | Master Mix BioU (12.2X) | Master Mix Dual (14.2X) |
Primer 2 (10 uM) | 1 | 12.2 | 0 | 14.2 |
BioU Primer (10 uM) | 1 | 0 | 12.2 | 14.2 |
2X Kapa SYBR Master Mix | 20 | 244 | 244 | 284 |
nfH2O | 16 | 195.2 | 195.2 | 213 |
Total* (+ 5 uL sample=40) | 35 | 451.4 | 451.4 | 525.4 |