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==== 2nd QUCIK RUN PCR to monitor cycle number ==== * PCR fail in the 1st run. I suspect it should be evaporation problem. However, I still saw primer dimer bands and the unexpected band ~125bp, which is not what I want. This meant PCR was actually100% fail, but why I did not see the expected 300bp band? * I a little doubt about the primers. It should be STD primers used to amplify the libraries prepared by Illumina adaptor. I decide to use the amplification primers I used to amplify the libraries (TruS F/R). I will use the same primer concentration as I used to amplify the library at 0.3uM instead of 0.5uM. * I will increase total reaction to be larger (12.5ul) and close the lid very tight. '''Components''' '''Volume (ul)''' KAPA HiFi HotStart ReadyMix (2X) 50.00 TruS F/R (10uM) 3.00 --> Final conc. of primers after adding template = 0.3uM H2O 7.00 '''Total''' '''60.00''' # Aliquot 7.5ul + 5ul bead-bound captured DNA (total 12.5ul) 2X # Ran PCR at 16 and 17 cycles on different thermocycler. '''LM-PCR program''' 98C, 45s --> [98C, 15s -> 60C, 30s -> 72C, 30s] 16X, 17X --> 72C, 1min --> Hold @8C * I still observe '''evaporation''' on one of the reaction ran at 16cycles, but not the one at 17cycles. * It's possible that the lid was losen by itself. * I MAY NEED TO BE VERY CAREFUL WHEN I INCUBATE SMALL VOLUME REACTION ON THERMOCYCLER * I continue to verify PCR product in 6% TBE gel by loading 5ul PCR product [[File:ZhangLab_2 2014-11-07 16hr 34min_TEST_17X-16X-SeqCapNor-Canc.jpg| 350px]] N = normal control sample C = cancer patient sample * This time, I observe the expected band at 300bp. YES!!!. This meant that the capture worked well. The signal of 16cycles as recommended by the User Guide looked a little faint while the signal of 17 cycles was very saturated. I think that the amplification of 16-cycle reaction was not very efficient because of the evaporation more than 50% of total volume. * I continued to amplify the rest of the bead-bound captured DNA (~42ul left)
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