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== Amplification == * I reduced extension time to 45 sec, and increased annealing temperature to 65C {| class="wikitable" style="text-align:center; | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' | align="center" style="background:#f0f0f0;"|'''9.5 rxn mix''' |- | adapter ligated DNA||20.00||0.00 |- | PCR_F (NEB, 10uM)||1.00||9.50 |- | NEBNext Index (10uM) ||1.00||0.00 |- | 2X KAPA SYBR MM||25.00||237.50 |- | H2O||3.00||28.50 |- | Total volume||50.00||475.00 |} * Aliquot 29ul, add 1ul of NEBNext Indx, 20ul of adapter ligated DNA {| {{table}} | align="center" style="background:#f0f0f0;"|'''Samples''' | align="center" style="background:#f0f0f0;"|'''Indx''' |- | 1ng UDG/Endo IV -1||NEBNext Index1 |- | 1ng UDG/Endo IV -2||NEBNext Index2 |- | 1ng UDG/Endo IV -3||NEBNext Index3 |- | 10ng UDG/Endo IV -1||NEBNext Index4 |- | 10ng UDG/Endo IV -2||NEBNext Index5 |- | 10ng UDG/Endo IV -3||NEBNext Index6 |- | 10ng USER/PNK -1||NEBNext Index7 |- | 10ng USER/PNK -2||NEBNext Index8 |- | NTC||NEBNext Index1 |} '''Program (Eppendorf Realplex)''' 98C - 30s, (98C - 10s, 65C - 30s, 72C - 45s)x15, 72C - 2min, hold at 15C [[File:20130430_lowinput_CGI.png| 650px]] * 1ng UDG/Endo IV : 13 cycles * 10ng UDG/Endo IV : 10 cycles * 10ng USER/PNK : 12 cycles * 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 2013-04-30 19hr 43min_CGI_qPCR.jpg| 400px]] * From the gel image, using NEB loop adaptor can remove most of the background from adaptor. The library size was reduced as expected since Jeff increased dUTP and got more amplification with smaller number of cycle.
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