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== Amplification == {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' | align="center" style="background:#f0f0f0;"|'''8.5 rxn mix''' |- | adapter ligated DNA||15.00||0.00 |- | PCR_F(10uM)||1.00||8.50 |- | PCR_R.N2IndX(10uM)||1.00||0.00 |- | H2O||8.00||68.00 |- | 2X KAPA SYBR MM||25.00||212.50 |- | Total volume||50.00||425.00 |} * Aliquot 34ul, add 1ul of PCR_R.N2Indx, 15ul of adapter ligated DNA {| {{table}} | align="center" style="background:#f0f0f0;"|'''Samples''' | align="center" style="background:#f0f0f0;"|'''Indx''' |- | 1ng_200bp||PCR_R.N2Indx1 |- | 2.5ng_200bp||PCR_R.N2Indx2 |- | 5ng_200bp||PCR_R.N2Indx3 |- | 1ng_400bp||PCR_R.N2Indx4 |- | 2.5ng_400bp||PCR_R.N2Indx5 |- | 5ng_400bp||PCR_R.N2Indx6 |- | NTC1 with adapters||PCR_R.N2Indx7 |- | NTC2 no adapters||PCR_R.N2Indx8 |} '''Program (Eppendorf Realplex)''' 98C - 30s, (98C - 10s, 62C - 30s, 72C - 30s)x20, 72C - 2min, hold at 15C * I set up total 20 cycles, but monitored to stop at any number of cycle before reaching a plateau * Stop at '''13 cycles''' '''qPCR curve''' [[File:20130424_qPCR_LowInpu.png |550px]] * PAGE analysis to verify the PCR products by loading 5 ul of PCR product without bead purification (1/10 of total volume) [[File:20130424_qPCR_LowInput.png| 330px]] * From the qPCR curve and gel image, the background from adapters was very high comparing to the signal of expected bands. The qPCR curves between NTC and experiment were very close for very low input. Verification by PAGE telling more information * 2.5ng and 5ng clearly showed amplification. The background should be clean after bead purification. Still did not see amplification for 1ng sample either 200bp or 400bp *These conditions show more improvement comparing to previous experiment with 20 cycles. * No significant different between 200bp and 400bp DNA fragments. Seem like 400bp show slightly stronger signal. * The signal was lower than Rui with 15 cycles amplification. Rui's result showed stronger signal than background. I might optimize annealing temperature by using two step PCRs to minimize the background form adapters. * The amount of unligated adapters after ligation reaction interfere qPCR curve. * Continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2013-4-25
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