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==== QUCIK RUN PCR to monitor cycle number ==== * The User Guide suggest the actual condition for amplification of captured DNA. However, this is my 1st time doing this experiment. I may have lower efficiency of DNA capture. I will do PCR -1 & +1 of the recommendation cycle number (16X). '''Components''' '''Volume (ul)''' KAPA HiFi HotStart ReadyMix (2X) 25.00 Post-LM-PCR Oligos 1&2 (5uM) 5.00 '''Total''' '''30.00''' # Aliquot 7.5ul + 5ul bead-bound captured DNA (total 12.5ul). # I include NTC of PCR by adding 5ul of H2O # Split 4ul 3X '''LM-PCR program''' 98C, 45s --> [98C, 15s -> 60C, 30s -> 72C, 30s] 15X, 16X, 17X --> 72C, 1min --> Hold @8C * I used thermocycler as including the beads in PCR will interfere fluorescent reading. * After taking PCR tube from thermocycler, I noticed evaporation. This might be because, I used too small volume and did not close the lid tight enough. I never had evaporation problem when I used Realplex qPCR even I set up reaction as small as 5ul. * I verify all PCR product in 6% TBE gel. I was not very sure that evaporation will cause a big problem. [[File:ZhangLab_2 2014-11-07 15hr 22min_TEST_15-17X-SeqCapNor-Canc.jpg| 480px]] N = normal control sample C = cancer patient sample
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