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=Bisulfite conversion = * EZ-96 DNA Methylation-Gold Kit * Catalog# D5007 (Shallow-Well Format)<br> *'''Reagent list''' - '''CT conversion reagent:''' to the CT conversion reagent bottle, add 9mL H2O, 500ul M-dissolving buffer, and 3mL M-dilution buffer, mix at RT with frequent vortexing or shaking for 15min<br> - '''M-Binding buffer'''<br> - '''M-Wash buffer:''' add 144mL of 100% EtOH to the 36mL M-Wash buffer (total volume = 180mL)<br> - '''M-desulphonation buffer'''<br> *'''Procedure''' # calculate 1.2ug vol. of DNA sample, and the vol. of H2O to adjust total volume to 20ul # add water to 96-conversion plate, then add DNA # add 130ul of CT conversion reagent to 20ul of DNA sample, mix sample by pipetting up and down # seal the plate with sticky film # Transfer the plate to a thermal cycler and perform the following steps. ## 98C for 10mim ## 64C for 2.5hr ## 4 hold (can be up to 20hr) # add 400ul of M-binding buffer to the well of the Silicon-A Binding plate mounted on a collection plate # transfer the samples from the conversion plate to the well of the Silicon-A Binding plate, mix by pipetting up and down # centrifuge at >3000xg for 5min, discard the flow through # add 400ul of M-Wash buffer to each well, centrifuge at >3000xg for 5 min # add 200ul of M-Desulphonation buffer to each well, incubate at RT (20C-30C) for 20min # centrifuge at >3000xg for 5min, discard the flow through # add 400ul of M-Wash buffer to each well, centrifuge at >3000xg for 5min, discard the flow through # add another 400ul of M-Wash buffer, centrifuge for 10 min # place the Silicon-A binding plate onto an Elution plate, add 15ul of M-Elution buffer directly to each well # incubate for 5min, centrifuge at >3000xg for 3min to elute the DNA # DNA samples are ready for the next analysis or store at -20C for later use * Note: for long term storage, store at or below -70C * Actual speed for centrifugation: 2,000rpm. *Note: The ID used for HIV samples will correspond to the position in the 96-well plate. {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Order''' | align="center" style="background:#f0f0f0;"|'''Sample ID in Zhang lab''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''volume for 1200ng (ul)''' | align="center" style="background:#f0f0f0;"|'''H20 (ul)''' | align="center" style="background:#f0f0f0;"|'''Total amount for bis-cvt (ng)''' | align="center" style="background:#f0f0f0;"|'''CT conversion reagent (ul)''' | align="center" style="background:#f0f0f0;"|'''Total volume (ul)''' |- | 1||A1||100||12||8||1200||130||150 |- | 2||A2||100||12||8||1200||130||150 |- | 3||A3||100||12||8||1200||130||150 |- | 4||A4||100||12||8||1200||130||150 |- | 5||A5||100||12||8||1200||130||150 |- | 6||A6||100||12||8||1200||130||150 |- | 7||A7||100||12||8||1200||130||150 |- | 8||A8||100||12||8||1200||130||150 |- | 9||A9||100||12||8||1200||130||150 |- | 10||A10||100||12||8||1200||130||150 |- | 11||A11||100||12||8||1200||130||150 |- | 12||A12||100||12||8||1200||130||150 |- | 13||B1||100||12||8||1200||130||150 |- | 14||B2||100||12||8||1200||130||150 |- | 15||B3||100||12||8||1200||130||150 |- | 16||B4||100||12||8||1200||130||150 |- | 17||B5||100||12||8||1200||130||150 |- | 18||B6||100||12||8||1200||130||150 |- | 19||B7||100||12||8||1200||130||150 |- | 20||B8||100||12||8||1200||130||150 |- | 21||B9||100||12||8||1200||130||150 |- | 22||B10||100||12||8||1200||130||150 |- | 23||B11||100||12||8||1200||130||150 |- | 24||B12||100||12||8||1200||130||150 |- | 25||C1||100||12||8||1200||130||150 |- | 26||C2||100||12||8||1200||130||150 |- | 27||C3||100||12||8||1200||130||150 |- | 28||C4||100||12||8||1200||130||150 |- | 29||C5||100||12||8||1200||130||150 |- | 30||C6||100||12||8||1200||130||150 |- | 31||C7||100||12||8||1200||130||150 |- | 32||C8||100||12||8||1200||130||150 |- | 33||C9||100||12||8||1200||130||150 |- | 34||C10||100||12||8||1200||130||150 |- | 35||C11||100||12||8||1200||130||150 |- | 36||C12||100||12||8||1200||130||150 |- | 37||D1||100||12||8||1200||130||150 |- | 38||D2||100||12||8||1200||130||150 |- | 39||D3||100||12||8||1200||130||150 |- | 40||N37-1||100||12||8||1200||130||150 |- | 41||N37-2||100||12||8||1200||130||150 |- | 42||N37-3||100||12||8||1200||130||150 |- | 43||N37-4||100||12||8||1200||130||150 |- | 44||N37-5||100||12||8||1200||130||150 |- | 45||N37-6||100||12||8||1200||130||150 |- | 46||N37-7||100||12||8||1200||130||150 |- | 47||N37-8||100||12||8||1200||130||150 |- | 48||N37-9||100||12||8||1200||130||150 |- | 49||N37-10||100||12||8||1200||130||150 |}<br> * Plate layout {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Plat layout''' | align="center" style="background:#f0f0f0;"|'''1''' | align="center" style="background:#f0f0f0;"|'''2''' | align="center" style="background:#f0f0f0;"|'''3''' | align="center" style="background:#f0f0f0;"|'''4''' | align="center" style="background:#f0f0f0;"|'''5''' | align="center" style="background:#f0f0f0;"|'''6''' | align="center" style="background:#f0f0f0;"|'''7''' | align="center" style="background:#f0f0f0;"|'''8''' | align="center" style="background:#f0f0f0;"|'''9''' | align="center" style="background:#f0f0f0;"|'''10''' | align="center" style="background:#f0f0f0;"|'''11''' | align="center" style="background:#f0f0f0;"|'''12''' |- | A '''A'''||A1||A2||A3||A4||A5||A6||A7||A8||A9||A10||A11||A12 |- | B|||||||||||||||||||||||| |- | C '''B'''||B1||B2||B3||B4||B5||B6||B7||B8||B9||B10||B11||B12 |- | D|||||||||||||||||||||||| |- | E '''C'''||C1||C2||C3||C4||C5||C6||C7||C8||C9||C10||C11||C12 |- | F|||||||||||||||||||||||| |- | G '''D'''||D1||D2||D3|||||||||||||||||| |- | H||N37-1 ||N37-2||N37-3||N37-4||N37-5||N37-6||N37-7||N37-8||N37-9||N37-10|||| |}<br> * 2011_06_01 == Qubit ss-DNA assay == * added 1ul of bis-cvt gDNA in total volume 200ul, so dilution factor = 200)<br> {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''sample ID''' | align="center" style="background:#f0f0f0;"|'''Conc. In Qubit (ng/mL)''' | align="center" style="background:#f0f0f0;"|'''Final conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="background:#f0f0f0;"|'''Yields (ng)''' |- | A1||184.00||36.80||15.00||552.00 |- | A2||316.00||63.20||15.00||948.00 |- | A3||208.00||41.60||15.00||624.00 |- | A4||224.00||44.80||15.00||672.00 |- | A5||176.00||35.20||15.00||528.00 |- | A6||218.00||43.60||15.00||654.00 |- | A7||240.00||48.00||15.00||720.00 |- | A8||220.00||44.00||15.00||660.00 |- | A9||365.00||73.00||15.00||1095.00 |- | A10||220.00||44.00||15.00||660.00 |- | A11||216.00||43.20||15.00||648.00 |- | A12||285.00||57.00||15.00||855.00 |- | B1||191.00||38.20||15.00||573.00 |- | B2||263.00||52.60||15.00||789.00 |- | B3||198.00||39.60||15.00||594.00 |- | B4||153.00||30.60||15.00||459.00 |- | B5||234.00||46.80||15.00||702.00 |- | B6||301.00||60.20||15.00||903.00 |- | B7||148.00||29.60||15.00||444.00 |- | B8||209.00||41.80||15.00||627.00 |- | B9||215.00||43.00||15.00||645.00 |- | B10||193.00||38.60||15.00||579.00 |- | B11||168.00||33.60||15.00||504.00 |- | B12||240.00||48.00||15.00||720.00 |- | C1||169.00||33.80||15.00||507.00 |- | C2||230.00||46.00||15.00||690.00 |- | C3||248.00||49.60||15.00||744.00 |- | C4||204.00||40.80||15.00||612.00 |- | C5||208.00||41.60||15.00||624.00 |- | C6||139.00||27.80||15.00||417.00 |- | C7||127.00||25.40||15.00||381.00 |- | C8||174.00||34.80||15.00||522.00 |- | C9||215.00||43.00||15.00||645.00 |- | C10||202.00||40.40||15.00||606.00 |- | C11||200.00||40.00||15.00||600.00 |- | C12||179.00||35.80||15.00||537.00 |- | D1||231.00||46.20||15.00||693.00 |- | D2||234.00||46.80||15.00||702.00 |- | D3||213.00||42.60||15.00||639.00 |- | N37-1||201.00||40.20||15.00||603.00 |- | N37-2||135.00||27.00||15.00||405.00 |- | N37-3||194.00||38.80||15.00||582.00 |- | N37-4||195.00||39.00||15.00||585.00 |- | N37-5||165.00||33.00||15.00||495.00 |- | N37-6||282.00||56.40||15.00||846.00 |- | N37-7||206.00||41.20||15.00||618.00 |- | N37-8||187.00||37.40||15.00||561.00 |- | N37-9||256.00||51.20||15.00||768.00 |- | N37-10||168.00||33.60||15.00||504.00 |}<br> == Capture set up == === Probe calculation === {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Subset ID''' | align="center" style="background:#f0f0f0;"|'''Adjusted Multiplier based on qPCR (Multiplier E)''' | align="center" style="background:#f0f0f0;"|'''Actual amount of probe required (probe:target ratio, 100:1)''' | align="center" style="background:#f0f0f0;"|'''Multiplier E x amount probe required''' | align="center" style="background:#f0f0f0;"|'''Probe concentration (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="background:#f0f0f0;"|'''Volume for 55rxn (ul)''' |- | DMR.s1||0.05||2.23||0.11||1.86||0.06||3.30 |- | DMR.s2||0.07||2.23||0.16||1.95||0.08||4.57 |- | DMR.s3||0.23||18.90||4.41||23.20||0.19||10.44 |- | DMR.s4||1.00||18.90||18.90||23.20||0.81||44.81 |- | DMR.s5||4.83||18.90||91.29||29.20||3.13||171.94 |- | DMR.Exp1||0.23||18.90||4.44||31.88||0.14||7.65 |- | DMR.Exp2||0.19||18.90||3.50||24.11||0.15||8.00 |- | DMR.Exp3||0.27||18.90||5.03||28.90||0.17||9.56 |- | 105nM Oligo suppressor||||||||||1.00||55.00 |- | ||||||||Total volume||5.73||315.28 |}<br> === Reaction mix === {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1rxn (ul)''' | align="center" style="background:#f0f0f0;"|'''55rxn mix (ul)''' |- | 10X AmpLigase Buffer||2.50||137.50 |- | DMR330k probe mix (ul)||4.73||260.15 |- | 105nM Oligo suppressor||1.00||55.00 |- | Total volume (ul)||8.23||452.65 |}<br> {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''sample ID''' | align="center" style="background:#f0f0f0;"|'''Final conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Volume for 400ng(ul)''' | align="center" style="background:#f0f0f0;"|'''H2O (ul)''' | align="center" style="background:#f0f0f0;"|'''Total amount of bis-cvt gDNA (ng)''' | align="center" style="background:#f0f0f0;"|'''Probe mix (ul)''' | align="center" style="background:#f0f0f0;"|'''10x Amligase buffer (ul)''' | align="center" style="background:#f0f0f0;"|'''105nM Oligo suppressor (ul)''' | align="center" style="background:#f0f0f0;"|'''Total volume (ul)''' |- | A1||36.80||10.87||5.90||400.00||4.73||2.50||1.00||25.00 |- | A2||63.20||6.33||10.44||400.00||4.73||2.50||1.00||25.00 |- | A3||41.60||9.62||7.15||400.00||4.73||2.50||1.00||25.00 |- | A4||44.80||8.93||7.84||400.00||4.73||2.50||1.00||25.00 |- | A5||35.20||11.36||5.41||400.00||4.73||2.50||1.00||25.00 |- | A6||43.60||9.17||7.60||400.00||4.73||2.50||1.00||25.00 |- | A7||48.00||8.33||8.44||400.00||4.73||2.50||1.00||25.00 |- | A8||44.00||9.09||7.68||400.00||4.73||2.50||1.00||25.00 |- | A9||73.00||5.48||11.29||400.00||4.73||2.50||1.00||25.00 |- | A10||44.00||9.09||7.68||400.00||4.73||2.50||1.00||25.00 |- | A11||43.20||9.26||7.51||400.00||4.73||2.50||1.00||25.00 |- | A12||57.00||7.02||9.75||400.00||4.73||2.50||1.00||25.00 |- | B1||38.20||10.47||6.30||400.00||4.73||2.50||1.00||25.00 |- | B2||52.60||7.60||9.17||400.00||4.73||2.50||1.00||25.00 |- | B3||39.60||10.10||6.67||400.00||4.73||2.50||1.00||25.00 |- | B4||30.60||13.07||3.70||400.00||4.73||2.50||1.00||25.00 |- | B5||46.80||8.55||8.22||400.00||4.73||2.50||1.00||25.00 |- | B6||60.20||6.64||10.13||400.00||4.73||2.50||1.00||25.00 |- | B7||29.60||13.51||3.26||400.00||4.73||2.50||1.00||25.00 |- | B8||41.80||9.57||7.20||400.00||4.73||2.50||1.00||25.00 |- | B9||43.00||9.30||7.47||400.00||4.73||2.50||1.00||25.00 |- | B10||38.60||10.36||6.41||400.00||4.73||2.50||1.00||25.00 |- | B11||33.60||11.90||4.87||400.00||4.73||2.50||1.00||25.00 |- | B12||48.00||8.33||8.44||400.00||4.73||2.50||1.00||25.00 |- | C1||33.80||11.83||4.94||400.00||4.73||2.50||1.00||25.00 |- | C2||46.00||8.70||8.07||400.00||4.73||2.50||1.00||25.00 |- | C3||49.60||8.06||8.71||400.00||4.73||2.50||1.00||25.00 |- | C4||40.80||9.80||6.97||400.00||4.73||2.50||1.00||25.00 |- | C5||41.60||9.62||7.15||400.00||4.73||2.50||1.00||25.00 |- | C6||27.80||14.39||2.38||400.00||4.73||2.50||1.00||25.00 |- | C7||25.40||15.75||1.02||400.00||4.73||2.50||1.00||25.00 |- | C8||34.80||11.49||5.28||400.00||4.73||2.50||1.00||25.00 |- | C9||43.00||9.30||7.47||400.00||4.73||2.50||1.00||25.00 |- | C10||40.40||9.90||6.87||400.00||4.73||2.50||1.00||25.00 |- | C11||40.00||10.00||6.77||400.00||4.73||2.50||1.00||25.00 |- | C12||35.80||11.17||5.60||400.00||4.73||2.50||1.00||25.00 |- | D1||46.20||8.66||8.11||400.00||4.73||2.50||1.00||25.00 |- | D2||46.80||8.55||8.22||400.00||4.73||2.50||1.00||25.00 |- | D3||42.60||9.39||7.38||400.00||4.73||2.50||1.00||25.00 |- | N37-1||40.20||9.95||6.82||400.00||4.73||2.50||1.00||25.00 |- | N37-2||27.00||14.81||1.96||400.00||4.73||2.50||1.00||25.00 |- | N37-3||38.80||10.31||6.46||400.00||4.73||2.50||1.00||25.00 |- | N37-4||39.00||10.26||6.51||400.00||4.73||2.50||1.00||25.00 |- | N37-5||33.00||12.12||4.65||400.00||4.73||2.50||1.00||25.00 |- | N37-6||56.40||7.09||9.68||400.00||4.73||2.50||1.00||25.00 |- | N37-7||41.20||9.71||7.06||400.00||4.73||2.50||1.00||25.00 |- | N37-8||37.40||10.70||6.07||400.00||4.73||2.50||1.00||25.00 |- | N37-9||51.20||7.81||8.96||400.00||4.73||2.50||1.00||25.00 |- | N37-10||33.60||11.90||4.87||400.00||4.73||2.50||1.00||25.00 |}<br> '''Program''' -> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h -> add 2.5ul SLN mix (2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 100uM dNTP) (2011_06_02) -> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold. (2011_06_03) === SLN mix === {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Stock conc.''' | align="center" style="background:#f0f0f0;"|'''Unit''' | align="center" style="background:#f0f0f0;"|'''Final conc.''' | align="center" style="background:#f0f0f0;"|'''Unit''' | align="center" style="background:#f0f0f0;"|'''Prepare volume 150''' |- | AmpliTaq Stoffel||10.00||U/ul||2.00||U/ul||30.00 |- | AmpLigase||5.00||U/ul||0.50||U/ul||15.00 |- | dNTP||1.00||mM||100.00||uM||15.00 |- | 10x AmpLigase Buffer||10.00||x||1.00||x||15.00 |- | H2O||||||||||75.00 |- | ||||||||Total||150.00 |}<br> * aliquot 15ul of SLN to strip tube, and add 2.5 ul to each tube. * Note: ** There was something wrong with sample in well A9-A12. The volume of reaction in these four well was lower than 10ul, but before capture the levels of the reaction in every well were the same. I think that it's possible that the 96-well plate was not sealed very well especially the border of the plate, so the evaporation possibly occurred. There was proof that the reagents were not missing added to these four wells since one of them could be amplified, but not in a good yield. In the future, there should be more concerns about the plate sealing. ** It would be better if the samples will be rearranged from A1-H1, ..., A2-H2,.... rather than A1-A12, B1-B12. This because the multichannel pipette has 8 channels, so it would be easier to add samples in the same column at once. In addition, it's less possibility to miss adding reagents in well No.9-12<br>
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