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== Amplification == {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' | align="center" style="background:#f0f0f0;"|'''13.5 rxn mix''' |- | adapter ligated DNA||15.00||0.00 |- | PCR_Fs(10uM)||1.00||13.50 |- | PCR_R.N2IndX(10uM)||1.00||0.00 |- | H2O||8.00||108.00 |- | 2X KAPA SYBR MM||25.00||337.50 |- | Total volume||50.00||675.00 |} * 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''' |- | 1. 1ng high/low? USER short incubation||PCR_R.N2Indx1 |- | 2. 1ng high/low? USER short incubation||PCR_R.N2Indx2 |- | 3. 10ng high/low? USER short incubation||PCR_R.N2Indx3 |- | 4. 10ng high/low? USER short incubation||PCR_R.N2Indx4 |- | 5. 1ng high/low? USER long incubation||PCR_R.N2Indx5 |- | 6. 1ng high/low? USER long incubation||PCR_R.N2Indx6 |- | 7. 10ng high/low? USER long incubation||PCR_R.N2Indx7 |- | 8. 10ng high/low? USER long incubation||PCR_R.N2Indx8 |- | 9. 1ng UDG||PCR_R.N2Indx21 |- | 10. 1ng UDG||PCR_R.N2Indx22 |- | 11. 10ng UDG||PCR_R.N2Indx23 |- | 12. 10ng UDG||PCR_R.N2Indx24 |- | 12. NTC||PCR_R.N2Indx24 |} '''Program (Eppendorf Realplex)''' 98C - 30s, (98C - 10s, 62C - 30s, 72C - 60s)x15, 72C - 2min, hold at 15C * Jeff suggested not go over 15 cycles. I stopped reaction for 10ng UDG at 13 cycles and the rest at 15 cycles to make sure that I would see amplification. '''qPCR curve''' [[File:20130425_lowinput_CGI.png| 650px]] * The qPCR curve of NTC was lower than other samples. This was not as usual since I expected to see high background of adapters especially in NTC. * PAGE analysis to verify the PCR products by loading 5 ul of PCR product without bead purification (1/10 of total volume) [[File:ZhangLab_2 2013-04-25 18hr 24min_lowinput_CGI_e.jpg| 520px]] [[File:ZhangLab_2 2013-04-25 18hr 16min_lowinput_CGI_e.jpg| 330px]] S and L: with USER UDG = Uracil DNA glycosylase * Will add more info * '''Comments from Dr. Zhang''' ** He suggested to use commercial adapter from NEB or HPLC purified adapter to avoid the effect of self ligated adapter due to the quality of adapter we used from IDT (desalted oligos). ** He suggested Jeff to increase the ratio od dUTP during MDA to minimize the size of library and tring USER with PNK to generate 3'-OH. ** Will be more details * Continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2013-4-30
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