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=== Preparing the LM-PCR === ==== recommendation conditions for 1rxn with 40ul captured DNA ==== '''Components''' '''Volume (ul)''' KAPA HiFi HotStart ReadyMix (2X) 50.00 Post-LM-PCR Oligos 1&2 (5uM) 10.00 --> Final conc. of primers after adding template = 0.5uM '''Total''' '''60.00''' # Pipette 30ul to 2X tube # Add 20ul of the bead-bound captured DNA to each tube. # Mix by vortexing # For NTC, add H2O ==== 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 ==== 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) ==== Amplification in a large volume ==== '''Components''' '''Volume (ul)''' '''2.2rxn (ul)''' KAPA HiFi HotStart ReadyMix (2X) 50.00 110.00 TruS F/R (10uM) 3.00 6.60 H2O 7.00 15.40 '''Total''' '''60.00''' # Aliquot 63ul + 42ul bead-bound captured DNA (total ul) # Split 52ul 2X '''LM-PCR program''' 98C, 45s --> [98C, 15s -> 60C, 30s -> 72C, 30s] 16X --> 72C, 1min --> Hold @8C :- Purified amplified captured DNA with 1X AMPure beads :- Elute with total volume 50ul of EB buffer :- Verify purified sequencing library in 6% TBE gel by loading 3ul. [[File:ZhangLab_2 2014-11-07 18hr 48min_beadpurified_UMRv1-SeqCap-Normal-cancer-3ul.jpg| 300px]] '''Library ID:''' 1 = NP-DNASMRT-Dec_SeqCapUMRv1-Oct29 2 = NP-SeqCap-pNormalSetA-Nov4 3 = NP-SeqCap-pCancerSetA-Nov4 * I am not sure if the upper background will interfere sequencing. I might overloaded DNA. Anyway,next time, I might reduce PCR cycle number to 15 cycles.
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