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== Amplification == === TEST PCR === {| {{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;"|'''6.5 rxn mix''' |- | Bis-cvt DNA||||||||||6.00||0.00 |- | 10X Reaction buffer||10||X||1||X||2.00||13.00 |- | dNTP mix||10||mM||0.25||mM||0.50||3.25 |- | TruS_F/R||10||uM||0.3||uM||0.60||3.90 |- | PfuTurbo Cx||2.5||Unit/ul||2.5||unit||0.40||2.60 |- | 50X SYBG||50||X||0.8||X||0.32||2.08 |- | H2O||||||||||10.18||66.17 |} :- 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 in 12% gel and run at 250 volts for 40min (running in regular 6% TBE gel at 250 volts, 25 min is okay) [[File:ZhangLab_2 2014-06-16 13hr 10min_CTT-RRBS-test15x-17X.jpg| 500px]] :- Determine # of cycles based on the intensity of the gel. For CTT-frozen samples, I would stop at 15X . For CTT-FFPE samples which have a faint smear, I would stopped at 17X. * Then set up reaction 100ul for each samples with 30ul BIS-DNA and split into 2X of 50ul to avoid bias from amplification (Ref. split 4X of 25ul) {| {{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;"|'''8.5 rxn mix''' |- | Bis-cvt DNA||||||||||15.00||0.00 |- | 10X Reaction buffer||10||X||1||X||5.00||42.50 |- | dNTP mix||10||mM||0.25||mM||1.25||10.63 |- | TruS_F/R||10||uM||0.3||uM||1.50||12.75 |- | PfuTurbo Cx||2.5||Unit/ul||2.5||unit||1.00||8.50 |- | 50X SYBG||50||X||0.8||X||0.80||6.80 |- | H2O||||||||||25.45||216.33 |} :- I did not include NTC as it clear in TEST PCR :- Aliquot 70 + 30ul DNA template :- Mix well. Split 2X of 50ul (actual ~48ul) :: ''95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X 18 cycles --> 72C for 2min'' :- Stopped at 15X for CTT-frozen and 17X for CTT-FFPE :- I took 5ul of PCR product from each tube and mix with 5ul of 2X loading buffer to verify that PCR work (even PCR curve looked fine) :- I pooled the two PCR replicatea (total ~87ul for each samples) and purified with 1x volume AMPure beads and eluted with 50ul EB buffer :- I did PAGE quantification by loading 4ul of bead-purified sequencing libraries [[File:ZhangLab_2 2014-06-16 17hr 52min_CTT-RRBS_100ng_PQ.jpg| 500px]] ==== PAGE quantification results ==== {| {{table}} class = wikitable | align="center" style="background:#f0f0f0;"|'''Sample''' | align="center" style="width:80px;background:#f0f0f0;"|'''TruSeq Index''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Amount in 45ul (ng)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 35ng (ul)''' |- | CTT-frozen-100ng_1||Ind_18||4.80||215.85||7.30 |- | CTT-frozen-100ng_2||Ind_19||4.64||208.89||7.54 |- | CTT-FFPE-100ng_1||Ind_20||1.05||47.31||33.29 |- | CTT-FFPE-100ng_2||Ind_21||1.77||79.63||19.78 |} :- I pooled 35ng each library based on PAGE quantification and did size selection in 2 well of 5-well 6% TBE gel [[File:ZhangLab_2 2014-06-17 14hr 06min_100ng_CTTRRBS_PAGE-SS.jpg| 350px]] :- I precipitated in 3tubes at -80C O/N and resuspended each tube with 11ul H2O --> total volume 33ul :- I took 4ul for PAGE verification. ==== PAGE verification ==== [[File:ZhangLab_2 2014-06-24 17hr 06min_RRBS_PAGE-verify.jpg| 500px]] '''Sequencing library ID: NP-RRBS-CTT-100ng, Jun13, 2014'''<br> - Length: 160-360bp<br> - Conc. by Qubit: XX ng/ul or XX nM
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