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=== PCR set up === {| {{table}} | align="center" style="background:#f0f0f0;"|'''DNA templates''' |- | 1. NTC |- | 2. DMR330k-SeqLib (GA sample) |- | 3. + control-0.1uM-no USER (100x diluted) |- | 4. Circular DNA |} {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1 rxn''' | align="center" style="background:#f0f0f0;"|'''4.2x rxn mix''' |- | DNA template||2.00||0.00 |- | 10 uM Syb_FP5A||1.00||4.20 |- | 10uM Syb_RP7||1.00||4.20 |- | 2x KAPA ||25.00||105.00 |- | H2O||21.00||88.20 |- | Total volume (ul)||50.00||210.00 |} * aliquot 48ul, add 2ul DNA template '''Program''' 98C 30s -> (98C 10s -> 55C 20s -> 72C 20s)x25-> 72C 3min->15C hold <br> [[File:qPCR-test-Syb-primers-20cycles-edit.png| 500px]]<br> * From the realtime-PCR curves, the second set primers can amplify both the sequencing library prepared by library-free primers and the amplicon amplified by randomly tagging primers for 50 cycles. This means that the second set primers works well. Next, I will test these primers with the DNA template amplified by randomly tagging primers for 2 cycles and after USER digestion. * In addition, there was no amplification in the reaction that contained only captured DNA. It confirmed that the primers amplified specifically to the DNA amplified by the first primer set not the circulalr DNA. * '''Note:''' ** Since this experiment was performed to test whether the primers work or not, the amount of DNA template in each reaction was not adjusted in the same amount.
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