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== Amplification == === TEST PCR === :- Prepare PCR in total 20ul reaction with 4 ul BIS-DNA {| {{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:60px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''10.5 rxn mix''' |- | Bis-cvt DNA||||||||||3.00||0.00 |- | 2X KAPA HiFi Uracil HotStart ReadyMix||2||X||1||X||5.00||52.50 |- | TruS_F/R||10||uM||0.3||uM||0.30||3.15 |- | 50X SYBG||50||X||0.4||X||0.08||0.84 |- | H2O||||||||||1.62||17.01 |- | Total||||||||||10.00|| |} :- Aliquot 3.5ul + 1.5ul BIS-DNA ::- ''98C for 45sec --> [98C for 15sec -> 60C for 30sec -> 72C for 30sec] X 13X --> 72C for 1min'' :- Verify all PCR product in 6% TBE gel [[File:ZhangLab_2 2014-12-12 20hr 24min_RRBS-DZ-5ul.jpg|500px]] * It looked like the pattern of size distribution was different from previous experiment as the size seemed to be more larger than sequencing library prepared from human tumor DNA. * I continued to amplified the rest of BIS-DNA in 100ul reaction with cycle number 14 cycles for sample C1, C2, H1, H3, 15 cycles for samples C4, H4, H5 and 16 cycles for sample C3 :- Purified with 1X AMPure bead, elute with 32ul EB buffer :- PAGE quantify by loading 4ul [[File:ZhangLab_2 2014-12-15 19hr 52min_PQ_SeqLib-4ul.jpg| 500px]] {| {{table}} border = 1 | align="center" style="width:100px;background:#f0f0f0;"|'''Sample IDs''' | align="center" style="width:100px;background:#f0f0f0;"|'''TruSeq Index''' | align="center" style="width:100px;background:#f0f0f0;"|'''Seq (RC)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Con. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Yield in 28ul''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 75ng (ul)''' |- | HC-C1||1.00||ATCACG||6.66||186.53||11.26 |- | HC-C2||2.00||CGATGT||7.07||198.07||10.60 |- | HC-C3||3.00||TTAGGC||3.16||88.38||23.76 |- | HC-C4||4.00||TGACCA||7.95||222.62||9.43 |- | HC-H1||5.00||ACAGTG||8.53||238.81||8.79 |- | HC-H2||6.00||GCCAAT||7.86||220.06||9.54 |- | HC-H3||7.00||CAGATC||9.33||261.24||8.04 |- | HC-H4||8.00||ACTTGA||7.78||217.90||9.64 |- | HC-H5||9.00||GATCAG||6.64||186.03||11.29 |}
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