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==== Quick test PCR to monitor PCR cycle number ==== * I use 1ul of bisulfite converted DNA in 10ul reaction for a quick test and will use this condition for large volume amplification after obtaining optimal PCR cycle number {| {{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;"|'''12X 10ul rxn''' |- | Adaptor ligated DNA||5.00||1.00||0.00 |- | 2X KAPA HiFi HotStart Uracil+ ReadyMix||25.00||5.00||60.00 |- | 10uM TruS F/R||1.50||0.30||3.60 |- | 50X SYBR||0.40||0.08||0.96 |- | H2O||18.10||3.62||43.44 |- | Total ||50.00||10.00|| |} :- Aliquot 9ul + 1ul template :- Mix well ::''98C for 45sec --> [98C for 15sec -> 60C for 30sec -> 72C for 30sec] X 8-18X --> 72C for 1min'' :- Load 3ul PCR product in 6% TBE gel to verify the amplified libraries [[File:ZhangLab_2 2014-12-05 16hr 57min_ILMN_qPCR-quicktest.jpg| 450px]] * Note: qPCR curves of KU_2 and KU_6 shoot up later than other samples with the same input amount. KU_2 curve was very close to NTC, but it had good amplification at 18 cycles. The color (orange) appeared in this sample may interfere fluorescence reading.
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