Daniel:Notebook/PosSequencing/2017-6-27
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emPCR 6 (Started Yesterday)[edit]
Protocol[edit]
- Prepare Water saturated compounds
- Mix 5 mL diethyl ether and 5 mL ddH2O in a falcon tube; allow to settle, extract top (ether) phase
- Mix 5 mL ethyl acetate and 5 mL ddH2O in a falcon tube; allow to settle, extract top (acetate) phase
- Emulsion Breaking
- With a 200 uL pipette add 100 uL diethyl ether to each sample; save the tip
- Adjust tip to 200 and using same tip pool samples in a new 1.5 mL tube
- Vortex the pooled sample in the 1.5 mL tube for 30 seconds
- Spin down the sample 13,000 rpm for 5 minutes
- Take off oil-DE mix (top layer) but do not disrupt the oil/aqueous interface
- Add 1000 uL ethyl acetate and vortex for 30 seconds
- Spin down sample at 13,000rpm for 3 minutes
- Remove ethyl acetate without disturbing the pellet
- Add 1000 uL diethyl ether and vortex for 30 seconds
- Spin down sample at 13,000rpm for 3 minutes
- Remove DE without disturbing the aqueous phase
- Allow DE to dry in the fume hood; resuspend beads in the solution and take 15 uL aside for testing
- 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 37 uL master mix to appropriate lanes
- Add 3 uL sample according to plate layout below File:PlateLayout20170627-emPCR6-SinglePrimer.png
- Run the following thermocycler program
- 95C 3 min
- 95C 3 sec
- 45C 30 sec
- 72C 20 sec
- plate read
- goto b x120
- 72C 2 min
- 16C hold
******
Reagent | uL in Single Rxn | Master Mix P2 (6.2X) | Master Mix BioU (6.2X) | Master Mix Dual (8.2X) |
Primer 2 (10 uM) | 1 | 6.2 | 0 | 8.2 |
BioU Primer (10 uM) | 1 | 0 | 6.2 | 8.2 |
2X Kapa SYBR Master Mix | 20 | 124 | 124 | 164 |
nfH2O | 16 | 99.2 | 99.2 | 123 |
Total* (+ 5 uL sample=40) | 37 | 229.4 | 229.4 | 303.4 |
Results[edit]
- PlateLayout20170627-emPCR6-SinglePrimer.png
Plate layout
- EmPCR620170623.singleprimer-plateCTs-normed.png
Plate CTs
- EmPCR620170623.singleprimer-rawcurves.png
Raw curves
So I stopped this run early, because the NTC lane of the dual primer samples amplified prior to either of the single-stranded primer lanes. However, the dsDNA lanes for both the nM and pM samples amplified 5+ cycles prior to the NTC. So the emPCR actually worked and there is something on those beads! However, the single-stranded protocol took away too much DNA again, even though I dropped the pH to 11.5.