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== Amplification == * Repeat all procedure exactly the same as previous experiment on 2014-08-03 === 1st round PCR (fix 12 cycles) === {| {{table}} border =1 | align="center" style="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)''' |- | Bis-cvt DNA||||||||||30.00 |- | 10X Reaction buffer||10||X||1||X||5.00 |- | dNTP mix||10||mM||0.25||mM||1.25 |- | TruS_F/R||10||uM||0.3||uM||1.50 |- | 50X SYBG||50||X||0.4||X||0.40 |- | PfuTurbo Cx||2.5||Unit/ul||1||unit||1.00 |- | H2O||||||||||10.85 |- | Total ||||||||||50.00 |} :- Aliquot 20 + 30ul BIS-DNA template :- Mix well :: ''95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X 12 --> 72C for 2min'' :- Purify with 1.25X AMPure beads :- Elute with 42ul EB Buffer :- Continue to 2nd round PCR. No PAGE verification. === 2nd round PCR === === 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. === Qubit dsDNA HS quantification of WGBS libraries from normal control cfDNA === {| {{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:60px;background:#f0f0f0;"|'''Dilution''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Total amount (ng)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Used for prev. exp (ng)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Amount left (ng)''' | align="center" style="width:80px;background:#f0f0f0;"|'''> 400ng''' |- | NC-1||4.1||ng/mL||2||100||0.41||80.00||32.80||0.00||32.80||NO |- | NC-2||13.9||ng/mL||2||100||1.39||80.00||111.20||0.00||111.20||NO |- | NC-3||18||ng/mL||2||100||1.80||80.00||144.00||0.00||144.00||NO |- | NC-5||4.71||ng/mL||2||100||0.47||80.00||37.68||0.00||37.68||NO |- | NC-6||42.5||ng/mL||2||100||4.25||80.00||340.00||100.00||240.00||NO |- | NC-7||4.69||ng/mL||2||100||0.47||80.00||37.52||0.00||37.52||NO |- | NC-8||15.2||ng/mL||2||100||1.52||80.00||121.60||0.00||121.60||NO |- | NC-9||37.2||ng/mL||2||100||3.72||80.00||297.60||100.00||197.60||NO |- | NC-12||29.9||ng/mL||2||100||2.99||80.00||239.20||0.00||239.20||NO |- | NC-13||16.4||ng/mL||2||100||1.64||80.00||131.20||0.00||131.20||NO |- | NC-14||12.8||ng/mL||2||100||1.28||80.00||102.40||0.00||102.40||NO |- | NC-15||12.9||ng/mL||2||100||1.29||80.00||103.20||0.00||103.20||NO |- | NC-16||13.3||ng/mL||2||100||1.33||80.00||106.40||0.00||106.40||NO |- | NC-17||38.6||ng/mL||2||100||3.86||80.00||308.80||0.00||308.80||NO |- | NC-18||26.3||ng/mL||2||100||2.63||80.00||210.40||0.00||210.40||NO |- | NC-19||4.74||ng/mL||2||100||0.47||80.00||37.92||0.00||37.92||NO |- | NC-20||3.96||ng/mL||2||100||0.40||80.00||31.68||0.00||31.68||NO |- | NC-21||4.13||ng/mL||2||100||0.41||80.00||33.04||0.00||33.04||NO |- | NC-22||27.1||ng/mL||2||100||2.71||80.00||216.80||0.00||216.80||NO |- | NC-23||17.6||ng/mL||2||100||1.76||80.00||140.80||0.00||140.80||NO |- | NC-24||3.87||ng/mL||2||100||0.39||80.00||30.96||0.00||30.96||NO |- | NC-27||68.8||ng/mL||2||100||6.88||80.00||550.40||100.00||450.40||YES |- | NC-29||35.9||ng/mL||2||100||3.59||80.00||287.20||0.00||287.20||NO |- | NC-30||64.6||ng/mL||2||100||6.46||80.00||516.80||100.00||416.80||YES |}
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