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Daniel:Notebook/PosSequencing/2017-6-23
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=Beadless emPCR (Started [[Daniel:Notebook/PosSequencing/2017-6-22|Yesterday]])= [[Daniel:Notebook/PosSequencing|Back to calendar]] ==Protocol== <ol start="5"> <li>Prepare Water saturated compounds</li> <ol type="A"> <li>Mix 5 mL diethyl ether and 5 mL ddH2O in a falcon tube; allow to settle, extract top (ether) phase</li> <li>Mix 5 mL ethyl acetate and 5 mL ddH2O in a falcon tube; allow to settle, extract top (acetate) phase</li> </ol> <li>Emulsion Breaking</li> <ol type="A"> <li>With a 200 uL pipette add 100 uL diethyl ether to each sample; save the tip</li> <li>Adjust tip to 200 and using same tip pool samples in a new 1.5 mL tube</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-DE mix (top layer) but do not disrupt the oil/aqueous interface</li> <li>Add 1000 uL ethyl acetate and vortex for 30 seconds</li> <li>Spin down sample at 13,000rpm for 3 minutes</li> <li>Remove ethyl acetate without disturbing the pellet</li> <li>Add 1000 uL diethyl ether and vortex for 30 seconds</li> <li>Spin down sample at 13,000rpm for 3 minutes</li> <li>Remove DE without disturbing the aqueous phase</li> <li>Allow DE to dry in the fume hood</li> </ol> ****** <li>Bead Capture</li> <ol type="A"> <li>Add equal volume (100 uL) Dynabuffer wash buffer and 5 uL beads to 1.5 mL tube and vortex for 30 seconds</li> <li>Incubate at RT for 15 minutes</li> <li>Pull down with magnet; remove supernatant; <span style="background:#FFFF00">Save supernatant for later</span></li> <li>Wash twice with 100 uL dynabuffer</li> <li>Resuspend in 50 uL dynabuffer</li> </ol> ****** <li>qPCR</li> <ol type="A"> <li>Make the following 10.2X (1X) master mix</li> <ol type="a"> <li>10.2 (1) uL 10 uM 2Biotin Uracil</li> <li>10.2 (1) uL 10 uM Primer 2</li> <li>204 (20) uL Kapa SYBR Fast 2X master mix</li> <li>163.2 (16) uL nfH<sub>2</sub>O</li></ol> <li>Aliquot 38 uL master mix to appropriate lanes</li> <li>Add 2 uL sample according to plate layout below</li> [[Image:PlateLayout-20170623-BeadqPCR.png|500x350px]] <li>Run the following thermocycler program</li> <ol type="a"> <li>95C 3 min</li> <li>95C 3 sec</li> <li>45C 30 sec</li> <li>72C 20 sec</li> <li>plate read</li> <li>goto b x90</li> <li>72C 2 min</li> <li>16C hold</li> </ol></ol> </ol> ==Results== <gallery perrow=2 heights=250px widths=350px mode=packed-hover> File:PlateLayout-20170623-BeadqPCR.png|Plate Layout File:Beadless-emPCR-20170623-plateCTs-normed.png|Plate CTs File:Beadless-emPCR-20170623-rawcurves.png|Raw Curves </gallery> 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. [[Category:PosSeq]] [[Category:20170622]]
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