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== <span style="color:blue">Part I: Re-amplification of WGBS libraries of normal plasma samples</span> == [[Noi/NOTES/2014-8-15|'''Link to WGBS library prep of normal plasma sample''']] * Most of normal plasma WGBS libraries have very low yield. I need to re-amplify to increase the yield and have some left for future experiment. * I did a test on few samples, including NC-7, NC-7, and NC-30, for number of cycle to re-amplify . '''Template amount for 30ul run''' {| {{table}} border = 1 | align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 3.6ng''' | align="center" style="width:80px;background:#f0f0f0;"|'''H2O to 10ul''' |- | NC-7||0.47||7.66||2.34 |- | NC-8||1.52||2.37||7.63 |- | NC-30||6.46||0.56||9.44 |} '''PCR Mix''' {| {{table}} border = 1 | align="center" style="width:180px;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;"|'''3.5 rxn mix''' |- | Purified 1st round DNA||||||||||10.00||0.00 |- | 5X Phusion HF buffer||5||X||1||X||6.00||21.00 |- | dNTP mix||10||mM||0.25||mM||0.75||2.63 |- | TruS_F/R||10||uM||0.3||uM||0.90||3.15 |- | 50X SYBR||50||X||0.4||X||0.24||0.84 |- | Phusion HF||2||unit/ul||||||0.30||1.05 |- | H2O||||||||||11.81||41.34 |- | Total||||||||||30.00|| |} :- Aliquot 20ul + DNA template with adjusted H2O total 10ul :- Split 9ul 3X ::98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X 5,6,7 cycles --> 72C for 2min :- Added 2ul of 6X loading dye :- Loaded 6ul of PCR and dye mix in 6% TBE gel [[File:ZhangLab_2 2014-11-03 13hr 46min_test-reamp-wgbs.jpg| 400px]] * 6 cycles seemed to be over-amplified either based on gel image or qPCR curve (not shown). I will use the same PCR condition to amplify the rest of libraries with the same template concentration at 5 cycles. {| {{table}} border = 1 | align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 30ng (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:80px;background:#f0f0f0;"|'''H2O to 80 ul''' |- | NC-1||0.41||73.17||NC-1||6.83 |- | NC-2||1.39||21.58||NC-2||58.42 |- | NC-3||1.8||16.67||NC-3||63.33 |- | NC-5||0.47||63.83||NC-5||16.17 |- | NC-6||4.25||7.06||NC-6||72.94 |- | NC-7||0.47||63.83||NC-7||16.17 |- | NC-8||1.52||19.74||NC-8||60.26 |- | NC-9||3.72||8.06||NC-9||71.94 |- | NC-12||2.99||10.03||NC-12||69.97 |- | NC-13||1.64||18.29||NC-13||61.71 |- | NC-14||1.28||23.44||NC-14||56.56 |- | NC-15||1.29||23.26||NC-15||56.74 |- | NC-16||1.33||22.56||NC-16||57.44 |- | NC-17||3.86||7.77||NC-17||72.23 |- | NC-18||2.63||11.41||NC-18||68.59 |- | NC-19||0.47||63.83||NC-19||16.17 |- | NC-20||0.4||75.00||NC-20||5.00 |- | NC-21||0.41||73.17||NC-21||6.83 |- | NC-22||2.71||11.07||NC-22||68.93 |- | NC-23||1.76||17.05||NC-23||62.95 |- | NC-24||0.39||76.92||NC-24||3.08 |- | NC-27||6.88||4.36||NC-27||75.64 |- | NC-29||3.59||8.36||NC-29||71.64 |- | NC-30||6.46||4.64||NC-30||75.36 |} '''PCR Mix''' {| {{table}} border =1 | align="center" style="width:180px;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;"|'''25rxn''' |- | Purified 1st round DNA||||||||||80.00||0.00 |- | 5X Phusion HF buffer||5||X||1||X||50.00||1,250.00 |- | dNTP mix||10||mM||0.25||mM||6.25||156.25 |- | TruS_F/R||10||uM||0.3||uM||7.50||187.50 |- | 50X SYBR||50||X||0.4||X||2.00||50.00 |- | Phusion HF||2||unit/ul||||||2.50||62.50 |- | H2O||||||||||101.75||2,543.75 |- | Total||||||||||250.00|| |} :- Aliquot 170ul to each tube + 80 ul of DNA template adjusted volume with H2O :- Split ~60ul 4X in 96-well plate ::98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X '''5''' cycles --> 72C for 2min :- Purified with 1X diluted AMPure bead (dilute 1:1 with 20% PEG/5N NaCl) :- Elute with 60ul EB Buffer. * NOTE: SeqCap experiment requires DNA resuspended in H2O not TE or EB buffer. However, in this case, I plan to pool the library and re-purify with 1 round of the bead agin to completely remove adaptor dimer background and resuspend the pooled libraries with small volume of H2O to reduce evaporation time that will be explained in SeqCap experiment. ==== Qubit dsDNA HS assay result ==== {| {{table}} border = 1 | align="center" style="width:120px;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.''' | align="center" style="width:60px;background:#f0f0f0;"|'''Unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Yield in 60ul''' |- | NC-1||56.4||ng/mL||1.5||133.3||56.4||ng/mL||7.52||451.20 |- | NC-2||53.9||ng/mL||1.5||133.3||53.9||ng/mL||7.19||431.20 |- | NC-3||58.6||ng/mL||1.5||133.3||58.6||ng/mL||7.81||468.80 |- | NC-5||59.5||ng/mL||1.5||133.3||59.5||ng/mL||7.93||476.00 |- | NC-6||49.3||ng/mL||1.5||133.3||49.3||ng/mL||6.57||394.40 |- | NC-7||60.5||ng/mL||1.5||133.3||60.5||ng/mL||8.07||484.00 |- | NC-8||43.2||ng/mL||1.5||133.3||43.2||ng/mL||5.76||345.60 |- | NC-9||49.6||ng/mL||1.5||133.3||49.6||ng/mL||6.61||396.80 |- | NC-12||34.9||ng/mL||1.5||133.3||34.9||ng/mL||4.65||279.20 |- | NC-13||40.7||ng/mL||1.5||133.3||40.7||ng/mL||5.43||325.60 |- | NC-14||34.9||ng/mL||1.5||133.3||34.9||ng/mL||4.65||279.20 |- | NC-15||42.6||ng/mL||1.5||133.3||42.6||ng/mL||5.68||340.80 |- | NC-16||39.6||ng/mL||1.5||133.3||39.6||ng/mL||5.28||316.80 |- | NC-17||42.7||ng/mL||1.5||133.3||42.7||ng/mL||5.69||341.60 |- | NC-18||37.2||ng/mL||1.5||133.3||37.2||ng/mL||4.96||297.60 |- | NC-19||44.5||ng/mL||1.5||133.3||44.5||ng/mL||5.93||356.00 |- | NC-20||48.8||ng/mL||1.5||133.3||48.8||ng/mL||6.51||390.40 |- | NC-21||51.1||ng/mL||1.5||133.3||51.1||ng/mL||6.81||408.80 |- | NC-22||36.6||ng/mL||1.5||133.3||36.6||ng/mL||4.88||292.80 |- | NC-23||41.5||ng/mL||1.5||133.3||41.5||ng/mL||5.53||332.00 |- | NC-24||49.4||ng/mL||1.5||133.3||49.4||ng/mL||6.59||395.20 |- | NC-27||39.5||ng/mL||1.5||133.3||39.5||ng/mL||5.27||316.00 |- | NC-29||41.8||ng/mL||1.5||133.3||41.8||ng/mL||5.57||334.40 |- | NC-30||31.9||ng/mL||1.5||133.3||31.9||ng/mL||4.25||255.20 |}<br> <br>
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