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=Emulsion PCR Test 2 (Started [[Daniel:Notebook/PosSequencing/2017-5-25|Thursday May 25]])= [[Daniel:Notebook/PosSequencing|Back to Calendar]] Continuing the protocol from [[Daniel:Notebook/PosSequencing/2017-5-25|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== <ol start="5"> <li>Emulsion Breaking</li> <ol type="A"> <li>With a 200 uL pipette pool the emulsion into a 1.5 mL tube; save the tip and put aside</li> <li>Add 100 uL isopropyl alcohol (IPA) to tube with 200 uL tip (use new tip)</li> <li>Draw up IPA with tip from part A, mix to resuspend tip</li> <li>Vortex the pooled sample in the 1.5 mL tube for 30 seconds</li> <li>Spin down the sample 13,000 rpm for 5 minutes</li> <li>Take off oil-IPA mix (top layer) but do not disrupt the oil/aqueous interface</li> <li>Add 1000 uL IPA and vortex for 30 seconds</li> <li>Spin down sample at 13,000rpm for 3 minutes</li> <li>Again remove IPA without disturbing the pellet</li> <li>Add 500 uL 4X SSC wash buffer to 1.5 mL tube and vortex for 30 seconds</li> <li>Go back and mix thoroughly by pipetting up and down; the pellet may be hard to resuspend</li> <li>Leave on 1.5mL mixer at 1300rpm 1 minute</li> <li>Spin down sample at 13,000rpm for 1 minute</li> <li>Remove all but 40uL wash buffer</li> <li>Repeat steps 11 to 15 two times (j to o)</li> </ol> ****** <li>Strand Separation (DNA Denaturation)</li> <ol type="A"> <li>Add 500 uL KOH buffer (pH 12.0) and leave on 1.5mL mixer at 1300rpm 1 minute</li> <li>Spin down sample at 13,000rpm for 1 minute</li> <li>Remove all but 40uL buffer</li> <li>Repeat KOH steps A-C once more</li> <li>Add 500 uL 4X SSC buffer (pH 12.0) and leave on 1.5mL mixer at 1300rpm 1 minute</li> <li>Spin down sample at 13,000rpm for 3 minutes</li> <li>Remove all but 10uL buffer</li> </ol> ****** <li>Fluorescent Oligo Binding (All samples)</li> <ol type="A"> <li>For samples 2-4, re-pool samples into single tube</li> <li>Put suspension on magnet</li> <li>Resuspend beads in 25 uL 4X SSC buffer</li> <li>Make the following dye hybridization buffer</li> <ol type="a"> <li>2 uL 10 uM dye (dcprobe6-488)</li> <li>15 uL formamide</li> <li>8 uL nfH<sub>2</sub>O</li></ol> <li>Add 25 uL fluorescent probe in 60% formamide to each sample</li> <li>Incubate at room temp in the dark for 30 min</li> <li>Wash once in 2X SSC</li> <li>Resuspend in 50 uL 2XSSC</li> </ol> </ol> [[Category:PosSeq]] [[Category:20170525]]
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