Daniel:Notebook/PosSequencing/2017-6-15

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Emulsion PCR Test-Dual Biotin (Started Yesterday

Back to Calendar

Protocol

  1. Emulsion Breaking
    1. Set aside 15 uL for qPCR
    2. With a 200 uL pipette pool the emulsion into a 1.5 mL tube; save the tip and put aside
    3. Add 100 uL isopropyl alcohol (IPA) to tube with 200 uL tip (use new tip)
    4. Draw up IPA with tip from part A, mix to resuspend tip
    5. Vortex the pooled sample in the 1.5 mL tube for 30 seconds
    6. Spin down the sample 13,000 rpm for 5 minutes
    7. Take off oil-IPA mix (top layer) but do not disrupt the oil/aqueous interface
    8. Add 1000 uL IPA and vortex for 30 seconds
    9. Spin down sample at 13,000rpm for 3 minutes
    10. Again remove IPA without disturbing the pellet
    11. Add 500 uL 4X SSC wash buffer to 1.5 mL tube and vortex for 30 seconds
    12. Go back and mix thoroughly by pipetting up and down; the pellet may be hard to resuspend
    13. Leave on 1.5mL mixer at 1300rpm 1 minute
    14. Spin down sample at 13,000rpm for 1 minute
    15. Remove all but 40uL wash buffer
    16. Repeat steps 11 to 15 two times (j to o)
    17. On last wash, take 15 uL (of 100) for qPCR
    ******
    
  2. Strand Separation (DNA Denaturation)
    1. Add 500 uL KOH buffer (pH 12.0) and leave on 1.5mL mixer at 1300rpm 1 minute
    2. Spin down sample at 13,000rpm for 1 minute
    3. Remove all but 40uL buffer
    4. Repeat KOH steps A-C once more
    5. Add 500 uL 4X SSC buffer (pH 12.0) and leave on 1.5mL mixer at 1300rpm 1 minute
    6. Spin down sample at 13,000rpm for 3 minutes
    7. Remove all but 10uL buffer
    8. Resuspend in 80 uL 2X SSC buffer
    9. Use this for final step of qPCR