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Dinh:Probes Prep May2015
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===Probes production (Day 1)=== * Reagents used: 1.5x96wells plate, 6,600 uL KAPA Sybr Fast 2XMM, 600 uL QIAEX II silica beads * Set up ~15 min + 30 min run / 96 reactions = 1.5 hr qPCR * PCR cleanup with QIAEXII = 1 hr (would be faster if I can use an ultra-centrifuge to spin down 15 mL tubes). * Used 1X2.0 mL tubes to prepare each reaction master mix: Reagent Volume KAPA 2X Sybr Fast 1,100 uL Nuclease free water 1,100 uL AP1V4 8.8 uL AP2V4 8.8 uL Template (10nM) 4.4 uL (template was prepared by Noi from starting probes mix). 95C 30s -> [ 95C 15s -> 55C 45s -> 60C 30s read plate -> 60C 10s]x20cycles -> 60C 1 min * I used the CFX96 to perform qPCR. The incubation time at each temperature was increased to account for the overhead volume (>50uL). * Subsets A,C,D appears to saturate at 14 cycles while B,E saturate at 15 [[File:460K_prep_May2015_A-E_CFX96.jpg | 600px]] * I used QIAEX II beads to purify the PCR reactions. Each 2,200 uL total reaction was split into 5x2.0 mL tube. Used 20 uL of QIAEX II beads + 3X QX1 buffer. Eluted each tube with 50 uL nuclease free water first, then added more water for serial elution. {| {{table}} | align="center" style="background:#f0f0f0;"|'''Subset''' | align="center" style="background:#f0f0f0;"|'''cycles''' | align="center" style="background:#f0f0f0;"|'''100 uL reactions''' | align="center" style="background:#f0f0f0;"|'''QIAEX II yield, Nanodrop (ng/uL)''' | align="center" style="background:#f0f0f0;"|'''QIAEX II volume (uL)''' | align="center" style="background:#f0f0f0;"|'''Total yield (ug)''' |- | A||15||22||153.2||220||33.704 |- | B||15||22||128.4||220||28.248 |- | C||15||22||155.5||220||34.21 |- | D||15||22||154||230||35.42 |- | E||14||44||124.6||473||58.9358 |} * I expected 33 ug on average from 22 reactions and got about the right amount. Using QIAEX II beads saved time instead of performing ethanol precipitation and Bioneer column purification.
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