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== Amplification == * In previous experiment, I worried about losing small ligated DNA ~200bp, so I used 1.25X AMPure beads to purify 1st round PCR. In this library prep, I want to generate WGBS library for BSPP capture. I want the inser size at least 200bp for capture, so I can remove all the ligate DNA below 200bp by using 1X AMPure beads. === 1st round PCR (fix 12 cycles) === {| {{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./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;"|'''6.5x rxn mix''' |- | Bis-cvt DNA||||||||||30.00||0.00 |- | 10X Reaction buffer||10||X||1||X||5.00||32.50 |- | dNTP mix||10||mM||0.25||mM||1.25||8.13 |- | TruS_F/R||10||uM||0.3||uM||1.50||9.75 |- | 50X SYBG||50||X||0.4||X||0.40||2.60 |- | PfuTurbo Cx||2.5||Unit/ul||1||unit||1.00||6.50 |- | H2O||||||||||10.85||70.53 |- | Total ||||||||||50.00|| |} * Aliquot 20ul + 30ul BIS-DNA * Mix well :95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X 12 cycles --> 72C for 2min<br> [[File:ZhangLab_2 2014-09-10 12hr 45min_1stPCR_cfDNA1-4_12cycles_2ulPCR.jpg| 350px]] ==== Discussion ==== * Based on this result, I think that in the future to generate RRBS library or WGBS from ctDNA ~5ng, I do not need to do 2nd round PCR. In stead of using all 30ul of bis adaptor-ligated DNA for 50ul PCR, I can reduce amount of template and increase few more PCR cycles to get more PCR yields. In some application that do not require hight amount of libraries, I can stop PCR between 12-15 cycle based on qPCR curve. To make more amount of libraries for some downstream experiments, using lower concentration of template and increasing PCR cycle should be good enough. This can cut off bead purification step and save time. I can also try this on 1ng input. * However, for this experiment, I ran the gel test in the same time of bead purification. I need to go to 2nd PCR, but with fewer cycle number compare to previous experiment. I reduce the amount of template to 20% in PCR instead of 30% from total ~40ul eluted DNA from 1st round PCR. === 2nd round PCR === ==== OPTIMIZE PCR cycle number ==== [[File:ZhangLab_2 2014-09-10 13hr 19min_2ndPCR_cfDNA_6-7cycles.jpg| 500px]] * It seemed to be too saturated for 6 and 7 cycles. I the tried 4 and 5 cycles and also reduce 1st DNA template in PCR reaction from 30% to 20% [[File:ZhangLab_2014-09-10_2ndPCR_cfDNA_4-5cycles_2ul-in-10.jpg| 500px]] === 2nd round PCR (large volume) === * From gel image above, the amplification for sample cf1 and cf4 were still saturated, so I ran 3 cycles for these two samples and 4 cycles for sample cf2 and cf3 in total 170ul (mix before split into 3x of 55ul) {| {{table}} border = 1 | align="center" style="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:80px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''6 rxn mix''' |- | Purified 1st round DNA||||||||||34.00|| |- | 5X Phusion HF buffer||5||X||1||X||34.00||204.00 |- | dNTP mix||10||mM||0.25||mM||4.25||25.50 |- | TruS_F/R||10||uM||0.3||uM||5.10||30.60 |- | 50X SYBR||50||X||0.4||X||1.36||8.16 |- | Phusion HF||2||unit/ul||||||1.70||10.20 |- | H2O||||||||||89.59||537.54 |- | Total||||||||||170.00|| |} :- Aliquot 136 + 34ul DNA template :- Mix well. Split 3X of 55ul for each condition ::''98C for 2min --> [98C for 10sec -> '''60C for 30sec''' -> 72C for 1min] X [cf1 &cf4: 3X, cf2 & cf3: 4 X] cycles --> 72C for 2min''<br> :- Perform one round of 1x volume AMPure bead purification because the intensity of adaptor dimer wwas not very high and elute in ~65ul EB buffer :- Verify libraries by loading 1ul in 6% TBE gel. The 4 samples of 6-P-2 - 6-P-5 WGBS libraries that will be included for BSPP capture were also verified. [[File:ZhangLab_2 2014-09-11 01hr 14min_PAGE-verify_cf1-4_6P-2-5.jpg| 500px]] :- Quantify concentration of each library by Qubit dsDNA HS assay using 1ul of library {| {{table}} border = 1 | align="center" style="width:80px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. in the Qubit''' | align="center" style="width:60px;background:#f0f0f0;"|'''Unit''' | align="center" style="width:60px;background:#f0f0f0;"|'''uL used''' | align="center" style="width:80px;background:#f0f0f0;"|'''Dilution''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Yield in 60ul (ng)''' |- | cf1||41.6||ng/mL||1||200||8.33||499.80 |- | cf2||25.4||ng/mL||1||200||5.07||304.20 |- | cf3||47.2||ng/mL||1||200||9.43||565.80 |- | cf4||48||ng/mL||1||200||9.60||576.00 |}
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