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Dinh:Protocols/Probes Prep Nov24
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==Step 1: PCR Amplification == Reaction Mix (100ul reaction volume): {| border="1" !Reagent !Final Conc. !Vol (1x) !Vol (8x) !Vol (100x) |- |ddH20 | |49.4ul |395ul |4940ul |- | 2x KAPA qPCR Master Mix |1x |50ul |400ul |5000ul |- |AP1V4IU (100uM) |0.3uM |0.3ul |2.4ul |30ul |- |AP2V4 (100uM) |0.3uM |0.3ul |2.4ul |30ul |- |Template CpG30k (20nM) |33pg |0.05ul |0.4ul |5ul |- !Total | |100.05ul |800.4ul |10005ul |} Program 94C - 30 s 94C - 3 s 60C - 30 s Plate Read Cycle to 2 for 29 more times 72C - 5 min 15C - forever end Protocol: Place KAPA 2x qPCR MM on ice. Add ddH20, primers, and template to 15ml tube (RNAase/DNAase free). Vortex & leave on ice for 2-3 min Prepare strip tubes on cooling rack Add KAPA 2x MM & vortex, shake down reagents, then pour into 55ml solution basin Use multichannel p-100 pipettor. Pipette 101ul to each tube. For last row, use p-200 to pipette 101ul to each tube. Place caps on strip tubes Spin pairs of strip tubes for 5-10 s to remove bubbles Prepare new run on Chromo4 RT-PCR machine, let it get to 94 C Pause program, place tubes in machine, and resume run 3. Stopped at 19 cycles.
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