Daniel:Notebook/PosSequencing/2017-6-15: Difference between revisions
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>Djacobse (Created page with "=Emulsion PCR Test-Dual Biotin (Started Yesterday= Back to Calendar ==Protocol== <ol start="5"...") |
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<li><span style="background:#FFFF00">Set aside 15 uL for qPCR</span></li> | <li><span style="background:#FFFF00">Set aside 15 uL for qPCR</span></li> | ||
<li>With a 200 uL pipette pool the emulsion into a 1.5 mL tube; save the tip and put aside</li> | <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 | <li>Add 100 uL diethyl ether (DE) to tube with 200 uL tip (use new tip)</li> | ||
<li>Draw up | <li>Draw up DE 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>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>Spin down the sample 13,000 rpm for 5 minutes</li> | ||
<li>Take off oil- | <li>Take off oil-DE mix (top layer) but do not disrupt the oil/aqueous interface</li> | ||
<li>Add 1000 uL | <li>Add 1000 uL DE and vortex for 30 seconds</li> | ||
<li>Spin down sample at 13,000rpm for 3 minutes</li> | <li>Spin down sample at 13,000rpm for 3 minutes</li> | ||
<li>Again remove IPA without disturbing the pellet</li> | <li>Again remove IPA without disturbing the pellet</li> |
Revision as of 22:10, 14 June 2017
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 diethyl ether (DE) to tube with 200 uL tip (use new tip)
- Draw up DE 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-DE mix (top layer) but do not disrupt the oil/aqueous interface
- Add 1000 uL DE 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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