Daniel:Notebook/PosSequencing/2017-6-26
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Beadless emPCR (Started Thursday 6/22/17)
Since the results on Friday showed viable product (at least in the 100 nM sample), I'm going to finish up that experiment with strand denaturation and single primer qPCR.
Protocol
- Strand Separation (DNA Denaturation)
- Add 500 uL KOH buffer (pH 12.0)
- Spin down sample at 13,000rpm for 1 minute
- Remove all but 40uL buffer
- Repeat KOH steps A-C once more
- Add 500 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:PlateLayout-Beadless-emPCR-singleprimer-20170626.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 (3.2X) | Master Mix BioU (3.2X) | Master Mix Dual (5.2X) |
Primer 2 (10 uM) | 1 | 3.2 | 0 | 5.2 |
BioU Primer (10 uM) | 1 | 0 | 3.2 | 5.2 |
2X Kapa SYBR Master Mix | 20 | 64 | 64 | 104 |
nfH2O | 16 | 51.2 | 51.2 | 78 |
Total* (+ 5 uL sample=40) | 35 | 118.4 | 118.4 | 192.4 |