Daniel:Notebook/PosSequencing/2017-6-23
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Beadless emPCR (Started Yesterday)[edit]
Protocol[edit]
- Prepare Water saturated compounds
- Mix 5 mL diethyl ether and 5 mL ddH2O in a falcon tube; allow to settle, extract top (ether) phase
- Mix 5 mL ethyl acetate and 5 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
- Allow DE to dry in the fume hood
- Bead Capture
- Add equal volume (100 uL) Dynabuffer wash buffer and 5 uL beads to 1.5 mL tube and vortex for 30 seconds
- Incubate at RT for 15 minutes
- Pull down with magnet; remove supernatant; Save supernatant for later
- Wash twice with 100 uL dynabuffer
- Resuspend in 50 uL dynabuffer
- qPCR
- Make the following 10.2X (1X) master mix
- 10.2 (1) uL 10 uM 2Biotin Uracil
- 10.2 (1) uL 10 uM Primer 2
- 204 (20) uL Kapa SYBR Fast 2X master mix
- 163.2 (16) uL nfH2O
- Aliquot 38 uL master mix to appropriate lanes
- Add 2 uL sample according to plate layout below File:PlateLayout-20170623-BeadqPCR.png
- Run the following thermocycler program
- 95C 3 min
- 95C 3 sec
- 45C 30 sec
- 72C 20 sec
- plate read
- goto b x90
- 72C 2 min
- 16C hold
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Results[edit]
- PlateLayout-20170623-BeadqPCR.png
Plate Layout
- Beadless-emPCR-20170623-plateCTs-normed.png
Plate CTs
- Beadless-emPCR-20170623-rawcurves.png
Raw Curves
So this shows that it works well. Notice that the nM input (5 nM reaction concentration, 500 fmol total input). Also note that I diluted the beads fraction 1:10 prior to qPCR, which would mean that the initial amount is even higher.