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== Amplification == * Minor changes: Since 1ng of DNA input was not fragmented even there is a fraction of small fragments, I increase 1st round PCR from 12 cycles to 14 cycles to increase the amplified template. * To avoid losing small fragments below 200bp, I purified 1st round PCR with 1.25X AMPure bead. I tried not to increase the beads up to 1.5X or 2X since I did not want to get more adaptor dimers contamination in the next round PCR. I should have tested doing single round PCR for ~15-20 cycles and see if I can get sequencing libraries without 2nd round PCR. === 1st round PCR (fix 14 cycles) === {| {{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./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;"|'''4.5 rxn mix''' |- | Bis-cvt DNA||||||||||30.00||0.00 |- | 10X Reaction buffer||10||X||1||X||5.00||22.50 |- | dNTP mix||10||mM||0.25||mM||1.25||5.63 |- | TruS_F/R||10||uM||0.3||uM||1.50||6.75 |- | 50X SYBG||50||X||0.4||X||0.40||1.80 |- | PfuTurbo Cx||2.5||Unit/ul||1||unit||1.00||4.50 |- | H2O||||||||||10.85||48.83 |- | Total ||||||||||50.00|| |} :- Aliquot 20 + 30ul BIS-DNA template :- Mix well :: ''95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X 14 cycles --> 72C for 2min'' :- Purify with 1.25X AMPure beads (incubate DNA with beads 30min, the rest steps are the same as previous experiment) :- Elute with 42ul EB Buffer :- Continue to 2nd round PCR. No PAGE verification. === 2nd round PCR === ==== OPTIMIZE PCR cycle number ==== {| {{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;"|'''17 rxn mix''' |- | Purified 1st round DNA||||||||||3.00||0.00 |- | 5X Phusion HF buffer||5||X||1||X||2.00||34.00 |- | dNTP mix||10||mM||0.25||mM||0.25||4.25 |- | TruS_F/R||10||uM||0.3||uM||0.30||5.10 |- | 50X SYBR||50||X||0.4||X||0.08||1.36 |- | Phusion HF||2||unit/ul||||||0.10||1.70 |- | H2O||||||||||4.27||72.59 |- | Total||||||||||10.00|| |} :- Aliquot 28 + 12ul DNA template :- Mix well. Split 4X of 9ul ::''98C for 2min --> [98C for 10sec -> '''60C for 30sec''' -> 72C for 1min] X [8X, 9X, 10X, 12X] cycles --> 72C for 2min''<br> :- Added 3ul 6X loading dye to each strip tube & mix well :- Load 6ul PCR/dye mix in 6% TBe gel and run at 250 volts for 21 min * I planned to optimize PCR cycle number between 10-16 cycles by doing PCR in small volume (~10ul). However, based on qPCR curves saturated vary quick at ~8x. so I stopped reaction at 8X, 9X, 10X, and 12X cycles. * It looked like I should test PCR cycles at lower cycle number. I repeat experiment with the same condition at 6X and 7X cycles. [[File:ZhangLab_2 2014-08-01_noMspIRRBS_6-7cycles.jpg|460px]] [[File:ZhangLab_2 2014-08-01_noMspIRRBS_8-9cycles.jpg| 500px]] [[File:ZhangLab_2 2014-08-01_noMspIRRBS_10-12cycles.jpg| 460px]] 1: PC-P-1 2: PC-P-2 3: NTC 4: NTC PCR
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