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=== Quick test === {| {{table}} border = 1 | align="center" style="width:140px;background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc''' | align="center" style="width:80px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Final conc.''' | align="center" style="width:60px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''60 rxn mix''' |- | Purified 1st round DNA||||||||||3.00||0.00 |- | 5X Phusion HF buffer||5||X||1||X||2.00||120.00 |- | dNTP mix||10||mM||0.25||mM||0.25||15.00 |- | TruS_F/R||10||uM||0.3||uM||0.30||18.00 |- | 50X SYBR||50||X||0.4||X||0.08||4.80 |- | Phusion HF||2||unit/ul||||||0.10||6.00 |- | H2O||||||||||4.27||256.20 |- | Total||||||||||10.00|| |} :- Aliquot 14 + 6ul DNA template :- Mix well. ::''98C for 2min --> [98C for 10sec -> '''60C for 30sec''' -> 72C for 1min] X 7 and 9 cycles --> 72C for 2min''<br> :- I originally want to do 8 and 10 cycles, but qPCR curve getting saturated ~7 cycles. I then stopped at 7 and 9 cycles. :- Added 2ul 6X loading dye to each strip tube & mix well :- Load 6ul PCR/dye mix in 6% gel and run at 250 volts for 22min. :- To save TBE gel, I loaded only 7 cycles. I planned to load 9 cycles when 7 cycles give very faint signal. [[File:ZhangLab_2 2014-08-05 13hr 58min_wgbs_PCP-7X.jpg| 500px]] [[File:ZhangLab_2 2014-08-05 13hr 53min_wgbs_6P-7X.jpg| 500px]] [[File:ZhangLab_2 2014-08-05 14hr 02min_wgbs_7P-7x.jpg| 500px]] * From gel image I decided to do 2nd round PCR in larger volume at 8 cycles as Dr. Zhang suggested (1st: 12x, 2nd: 8x) {| {{table}} border = 1 | align="center" style="width:140px;background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc''' | align="center" style="width:80px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Final conc.''' | align="center" style="width:60px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''24.5 rxn mix''' |- | Purified 1st round DNA||||||||||30.00||0.00 |- | 5X Phusion HF buffer||5||X||1||X||20.00||490.00 |- | dNTP mix||10||mM||0.25||mM||2.50||61.25 |- | TruS_F/R||10||uM||0.3||uM||3.00||73.50 |- | 50X SYBR||50||X||0.4||X||0.80||19.60 |- | Phusion HF||2||unit/ul||||||1.00||24.50 |- | H2O||||||||||42.70||1046.15 |- | Total||||||||||100.00|| |} :- Aliquot 70 + 30ul DNA template :- Mix well. ::''98C for 2min --> [98C for 10sec -> '''60C for 30sec''' -> 72C for 1min] X 8 cycles --> 72C for 2min''<br> :- I pool ~95ul PCR product from the two replicates and elute with 65ul EB buffer :- I did PAGE quantification by loading 4ul of bead-purified sequencing libraries [[File:ZhangLab_2 2014-08-05 17hr 52min_PQ_PC-P_4ul.jpg| 500px]] [[File:ZhangLab_2 2014-08-05 17hr 54min_PQ_6P-4ul.jpg| 490px]] [[File:ZhangLab_2 2014-08-05 17hr 50min_PQ_7P-4ul.jpg| 500px]] * Note that for this WGBS library prep from patient plasma DNA and previous experiment of RRBS, I only used dA:dC:dG for end repair. I forgot that there could be the end of fragmented DNA that is not digested by MspI, so I should include dT in end repair mix. EspecialIy for WGBS of small fragment DNA in plasma, I must include all 4 bases. I then have generated the new WGBS of above samples using all 4 dAdCdGdT(10:1:1:1 ratio) with minor change of sample list. * [[Noi/NOTES/2014-8-18| '''Continued on 2014-08-18''']]
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