Daniel:Notebook/PosSequencing/2017-5-30

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Emulsion PCR Test 2 (Started Thursday May 25)[edit]

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Continuing the protocol from Thursday. Note that this is a continuation after about 5 days so I don't know if that affected anything. If the results are poor I might want to consider repeating the experiment in a 1-2 day span, as it would be normally.

Protocol[edit]

  1. Emulsion Breaking
    1. With a 200 uL pipette pool the emulsion into a 1.5 mL tube; save the tip and put aside
    2. Add 100 uL isopropyl alcohol (IPA) to tube with 200 uL tip (use new tip)
    3. Draw up IPA with tip from part A, mix to resuspend tip
    4. Vortex the pooled sample in the 1.5 mL tube for 30 seconds
    5. Spin down the sample 13,000 rpm for 5 minutes
    6. Take off oil-IPA mix (top layer) but do not disrupt the oil/aqueous interface
    7. Add 1000 uL IPA and vortex for 30 seconds
    8. Spin down sample at 13,000rpm for 3 minutes
    9. Again remove IPA without disturbing the pellet
    10. Add 500 uL 4X SSC wash buffer to 1.5 mL tube and vortex for 30 seconds
    11. Go back and mix thoroughly by pipetting up and down; the pellet may be hard to resuspend
    12. Leave on 1.5mL mixer at 1300rpm 1 minute
    13. Spin down sample at 13,000rpm for 1 minute
    14. Remove all but 40uL wash buffer
    15. Repeat steps 11 to 15 two times (j to o)
    ******
    
  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
    ******
    
  3. Fluorescent Oligo Binding (All samples)
    1. For samples 2-4, re-pool samples into single tube
    2. Put suspension on magnet
    3. Resuspend beads in 25 uL 4X SSC buffer
    4. Make the following dye hybridization buffer
      1. 2 uL 10 uM dye (dcprobe6-488)
      2. 15 uL formamide
      3. 8 uL nfH2O
    5. Add 25 uL fluorescent probe in 60% formamide to each sample
    6. Incubate at room temp in the dark for 30 min
    7. Wash once in 2X SSC
    8. Resuspend in 50 uL 2XSSC