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Dinh:Protocols/Probes Prep Nov24
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==Notes:== 8x reaction mix was prepared as test run. 100ul was aliquoted to 6 tubes and amplified using RT-PCR for 26 cycles (over-amplified), amplification slope started at cycle 13 and plateaued at cycle 25. For the rest of the reaction mix, 25ul was aliquoted to 8 tubes. Only 4 tubes were amplified using the the same program for 26 cycles (over-amplified), amplification sloped started also at cycle 13 and plateaued at cycle 25. Concluded no difference between 25ul and 100ul reaction volumes. The leftover 4 tubes of 25ul reaction volume was used in another run: Program 95C - 30 s 95C - 3 s 60C - 30 s 72C - 3 s Plate Read Cycle to 2 for 29 more times 72C - 5 min 15C - forever end Recommended by KAPA qPCR manual: 95C denaturation (initital & in cycle), and 72C during plate read. However, for this run, amplification slope started at cycle 15 and plateaued also at cycle 23. Concluded that other program used was better suited for this probe set.
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