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=Aqueous PCR 2 (Started [[Daniel:Notebook/PosSequencing/2017-6-6|Tuesday]])= [[Daniel:Notebook/PosSequencing|Back to Calendar]] ==Protocol== <ol> <li>Supernatant Extraction</li> <ol type="A"> <li>Vortex qPCR lanes from [[Daniel:Notebook/PosSequencing/2017-6-6|Tuesday]] for several seconds</li> <li>Magnet pulldown for 1 minute; remove supernatant and keep, putting into new 0.2 mL tubes</li> </ol> <li>qPCR</li> <ol type="A"> <li>Make the following 6.2X (1X) master mix</li> <ol type="a"><li>142.6 (23) uL nfH<sub>2</sub>O</li> <li>6.2 (1) uL 10 uM Biotin-Uracil primer</li> <li>6.2 (1) uL 10 uM Primer 2</li> <li>155 (25) uL Kapa 2X SYBR master mix</li> </ol> <li>Add 50 uL (resuspend beads) to each of the 6 lanes from [[Daniel:Notebook/PosSequencing/2017-6-6|Tuesday]]'s qPCR following plate layout</li> [[Image:PlateLayout-20170609-aqPCRStage2.png|250x350px]] <li>Make the following 8.2X master mix</li> <ol type="a"><li>147.6 (18) uL nfH<sub>2</sub>O</li> <li>8.2 (1) uL 10 uM Biotin-Uracil primer</li> <li>8.2 (1) uL 10 uM Primer 2</li> <li>205 (25) uL Kapa 2X SYBR master mix</li> </ol> <li>Add 45 uL master mix to each of the H lanes following plate layout</li> <li>Add 5 uL appropriate supernatant, 1 pM barcode 2 oligo, or nfH<sub>2</sub>O following plate layout</li> <li>Apply caps tightly, vortex 5 seconds and spin down</li> <li>Use following cycles for qPCR</li> <ol type="a"> <li>95C 2 min</li> <li>95C 20 sec</li> <li>58C 30 sec</li> <li>70C 30 sec</li> <li>Goto b 40 times</li> <li>4C forever</li> </ol></ol> </ol> ==Results== <gallery perrow=2 heights=250px widths=350px mode=packed-hover> File:PlateLayout-20170609-aqPCRStage2.png|Plate Layout File:20170609-aqPCRStage2-plateCTs.png|Raw CT values File:20170609-aqPCRStage2-plateCTs-normed.png|CT values normalized to NTC (Lane A6) File:20170609-aqPCRStage2-rawcurves.png|Raw curves File:2017-06-09-aqPCR-beadFraction.png|Gel image-bead fraction File:2017-06-09-aqPCR-Supernatant.png|Gel image-supernatant fraction File:2017-06-09-aqPCR-beadFraction-BAKE.png|Gel image-bead fraction post 95C bake (12 min) </gallery> [[Category:PosSeq]] [[Category:20170606]]
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