Daniel:Notebook/PosSequencing/2017-6-20
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Emulsion PCR Test 5 (Started Yesterday)
Protocol
- Prepare Water saturated compounds
- Mix 3 mL diethyl ether and 3 mL ddH2O in a falcon tube; allow to settle, extract top (ether) phase
- Mix 3 mL ethyl acetate and 3 mL ddH2O in a falcon tube; allow to settle, extract top (acetate) phase
- Emulsion Breaking
- With a 200 uL pipette add 100 uL diethyl ether to each sample; save the tip
- Adjust tip to 200 and using same tip pool samples in a new 1.5 mL tube
- 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 ethyl acetate and vortex for 30 seconds
- Spin down sample at 13,000rpm for 3 minutes
- Remove ethyl acetate without disturbing the pellet
- Add 1000 uL diethyl ether and vortex for 30 seconds
- Spin down sample at 13,000rpm for 3 minutes
- Remove DE without disturbing the aqueous phase
- Add 500 uL 4X SSC wash buffer to 1.5 mL tube and vortex for 30 seconds
- Spin down sample at 13,000rpm for 1 minute
- Remove all but 40uL wash buffer
- Repeat steps 11 to 15 one more time (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)
- 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)
- Spin down sample at 13,000rpm for 1 minute
- Remove all but 10uL buffer
- Resuspend in 80 uL 2X SSC buffer
- Use this for final step of qPCR
- Single primer qPCR
- Make the following master mixes
- Aliquot 35 uL master mix to appropriate lanes
- Add 2 uL sample according to plate layout below [[Image:|522x348px]]
- Run the following thermocycler program
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x30
- 72C 2 min
- 16C hold
******
Results
- PlateLayout-SinglePrimer-DualBiotin-20170615.png
Plate layout
- Singleprimer-20170615-dualbiotin-plateCTs.png
Plate CTs
- Singleprimer-20170615-dualbiotin-plateCTs-normed.png
Plate CTs-normed
- Singleprimer-20170615-dualbiotin-rawcurves.png
Raw curves