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=== TEST PCR === {| {{table}} border = 1 | align="center" style="width:160px;background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc''' | align="center" style="width:60px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Final conc./amount''' | align="center" style="width:60px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''13 rxn mix''' |- | Bis-cvt DNA||||||||||6.00||0.00 |- | 10X Reaction buffer||10||X||1||X||2.00||26.00 |- | dNTP mix||10||mM||0.25||mM||0.50||6.50 |- | TruS_F/R||10||uM||0.3||uM||0.60||7.80 |- | 50X SYBG||50||X||0.8||X||0.32||4.16 |- | PfuTurbo Cx||2.5||Unit/ul||1||unit||0.40||5.20 |- | H2O||||||||||10.18||132.34 |- | Total ||||||||||20.00|| |} :- Aliquot 14 + 6ul DNA template :- Mix well. Split 2X of 9ul :: ''95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X 18 cycles --> 72C for 2min'' :- Stopped at 15X and 17X :- Added 10ul 2X loading dye to each strip tube & mix well :- Load 10ul PCR/dye mix (equivalent to 5ul PCR product) in 12% gel and run at 250 volts for 40min (running in regular 6% TBE gel at 250 volts, 25 min is okay) :- Determine # of cycles based on the intensity of the gel. If the smear between 160bp -360bp is too bright and has some background a long the lane, we can reduce number of cycles ~1-2x. If the smear is very faint, increase number of cycle ~1. Based on CTT-RRBS 100ng input experiment, I would allow PCR between 15-18 cycles. [[File:ZhangLab_2 2014-06-18 13hr 56min_PC-T_RRBS_15xTESTPCR.jpg| 490px]] [[File:ZhangLab_2 2014-06-18 13hr 56min_PC-T_RRBS_17xTESTPCR.jpg|500px]] * The intensity of 15X cycles is quite low compare to CTT-RRBS result (frozen tissue), which I use as the reference as the samples were derived from frozen tissue. At 17X, some samples have very faint band. Some of them look good. ---- ==== 2014-06-17 ==== * I do not want to go with too high number of cycles. I repeated experiment by doing everything almost the same as described above with minor changes. ** This time I increase spike in unmeth lambda DNA to 1% as scRRBS experiment. The reason is I could get only 1000 reads from 10M reads of spike-in DNA 0.01%. This is too low. I expected to get 100,000 reads from 1% spike-in DNA. ** Also, I used MethylCode kit from LifeTech as I am not sure that elution DNA 20ul (to fit volume for Zymo Lightning kit) may not sufficient to elute all DNA form AMPure beads after ligation reaction.<br> <br>
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