Daniel:Notebook/PosSequencing/2017-5-30
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Emulsion PCR Test 2 (Started Thursday May 25)[edit]
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]
- Emulsion Breaking
- 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)
- 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
- Fluorescent Oligo Binding (All samples)
- For samples 2-4, re-pool samples into single tube
- Put suspension on magnet
- Resuspend beads in 25 uL 4X SSC buffer
- Make the following dye hybridization buffer
- 2 uL 10 uM dye (dcprobe6-488)
- 15 uL formamide
- 8 uL nfH2O
- Add 25 uL fluorescent probe in 60% formamide to each sample
- Incubate at room temp in the dark for 30 min
- Wash once in 2X SSC
- Resuspend in 50 uL 2XSSC
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