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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;"|'''30 rxn mix''' |- | Purified 1st round DNA||||||||||3.00||0.00 |- | 5X Phusion HF buffer||5||X||1||X||2.00||60.00 |- | dNTP mix||10||mM||0.25||mM||0.25||7.50 |- | TruS_F/R||10||uM||0.3||uM||0.30||9.00 |- | 50X SYBR||50||X||0.4||X||0.08||2.40 |- | Phusion HF||2||unit/ul||||||0.10||3.00 |- | H2O||||||||||4.27||128.10 |- | Total||||||||||10.00|| |} :- Aliquot 7 + 3ul DNA template :- Mix well. ::''98C for 2min --> [98C for 10sec -> '''60C for 30sec''' -> 72C for 1min] X 8 cycles --> 72C for 2min''<br> :- 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. [[File:ZhangLab_2 2014-08-17 16hr 58min_test-2ndPCR_NC-8x_gel1.jpg|450px]] [[File:ZhangLab_2 2014-08-17 16hr 58min_test-2ndPCR_NC-8x_gel2.jpg|450px]] [[File:ZhangLab_2 2014-08-17 16hr 58min_test-2ndPCR_NC-8x_gel3.jpg|430px]] * From quick qPCR test, there are variation of the intensity of libraries size ~300bp. 8 cycles seem to be over-amplified for almost samples. There are some samples that had very faint intensity of the libraries. I need to varied PCR cycle number for some samples. {| {{table}} border = 1 | align="center" style="width:140px;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.''' | align="center" style="width:60px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''27 rxn mix''' |- | Purified 1st round DNA||||||||||36.00||0.00 |- | 5X Phusion HF buffer||5||X||1||X||24.00||648.00 |- | dNTP mix||10||mM||0.25||mM||3.00||81.00 |- | TruS_F/R||10||uM||0.3||uM||3.60||97.20 |- | 50X SYBR||50||X||0.4||X||0.96||25.92 |- | Phusion HF||2||unit/ul||||||1.20||32.40 |- | H2O||||||||||51.24||1383.48 |- | Total||||||||||120.00|| |} :- Aliquot 84 + 36ul DNA template :- Mix well. Split 38ul X3 ::''98C for 2min --> [98C for 10sec -> '''60C for 30sec''' -> 72C for 1min] X 7 cycles --> 72C for 2min''<br> :- I pool ~110ul PCR product from the two replicates, purify with 2 rounds of 1X AMPure beads (fist round elute with EB buffer 60ul. 2nd round elute with 65ul EB buffer. :- Loaded 2ul in 6% TBE gel for PAGE verification [[File:ZhangLab_2 2014-08-18 14hr 52min_PQ_wgbs_NC-2ul_gel1.jpg| 450px]] [[File:ZhangLab_2 2014-08-18 14hr 52min_PQ_wgbs_NC-2ul_gel2.jpg| 450px]] [[File:ZhangLab_2 2014-08-18 14hr 52min_PQ_wgbs_NC-2ul_gel3.jpg| 320px]] * Different from WGBS from cancer patient small fragment DNA, there are high variations of library amount among different samples as mentioned from quick qPCR. Also, the last two samples (NC-3 and NC-4) ligated with T4 DNA ligase left at RT almost 2 days had comparable result compare to the same sample ligated with proper stored T4 DNA ligase.
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