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== Amplification == * I prepared 2 tubes of PCR mix for primer pairs of TruSeq adaptor and a customized adaptor with UMI {| {{table}} border = 1 | align="center" style="width:240px;background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''1X 50ul rxn''' | align="center" style="width:80px;background:#f0f0f0;"|'''10ul rxn''' | align="center" style="width:80px;background:#f0f0f0;"|'''6 rxn''' |- | Adaptor ligated DNA||20.00||2.00||0.00 |- | 2X KAPA HiFi Hotstart ReadyMix||25.00||5.00||30.00 |- | 10uM TruS F/R or 10uM SG_PCR F/RInd1||1.50||0.30||1.80 |- | 50X SYBR||0.40||0.08||0.48 |- | H2O||3.10||2.62||15.72 |- | Total ||50.00||10.00|| |} * I used less of DNA template than the instruction :- Aliquot 8 + 2ul adaptor ligated DNA :- Mix well ::''98C for 45sec --> [98C for 15sec -> 60C for 30sec -> 72C for 30sec] X 10 --> 72C for 1min'' [[File:2014-11-27_qPCR-TruSeqAdaptor-10x.PNG|500px]] [[File:2014-11-27_qPCR-CustomUMIAdaptor-10x.PNG|500px]] :- Verify 4ul of PCR product in 6% TBE gel [[File:ZhangLab_2 2014-11-27 14hr 44min_KAPAHyper_TruS-CustomUMI-10x_v2.jpg|500px]] * Dr. Zhang thought that there should be something about the number of annealed adaptor molecules even I used the same concentration for annealing. The present of 125bp adaptor dimers in TruSeq adaptor but very faint in customized adaptor could also partially contribute to qPCR curve. * He suggested me to repeat experiment using one plasma DNA sample and use1X, 2X and 4X of customized adaptor. ----
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