Daniel:Notebook/PosSequencing/2017-6-15
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Emulsion PCR Test-Dual Biotin (Started Yesterday
Protocol
- Emulsion Breaking
- Set aside 15 uL for qPCR
- With a 200 uL pipette pool the emulsion into a 1.5 mL tube; save the tip and put aside
- Add 100 uL isopropyl alcohol (IPA) to tube with 200 uL tip (use new tip)
- Draw up IPA with tip from part A, mix to resuspend tip
- 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-IPA mix (top layer) but do not disrupt the oil/aqueous interface
- Add 1000 uL IPA and vortex for 30 seconds
- Spin down sample at 13,000rpm for 3 minutes
- Again remove IPA without disturbing the pellet
- Add 500 uL 4X SSC wash buffer to 1.5 mL tube and vortex for 30 seconds
- Go back and mix thoroughly by pipetting up and down; the pellet may be hard to resuspend
- Leave on 1.5mL mixer at 1300rpm 1 minute
- Spin down sample at 13,000rpm for 1 minute
- Remove all but 40uL wash buffer
- Repeat steps 11 to 15 two times (j to o)
- On last wash, take 15 uL (of 100) for qPCR
- Strand Separation (DNA Denaturation)
- Add 500 uL KOH buffer (pH 12.0) and leave on 1.5mL mixer at 1300rpm 1 minute
- 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) and leave on 1.5mL mixer at 1300rpm 1 minute
- Spin down sample at 13,000rpm for 3 minutes
- Remove all but 10uL buffer
- Resuspend in 80 uL 2X SSC buffer
- Use this for final step of qPCR
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