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= Repeat bisulfite conversion = '''Reagent prep''' Add the following reagents to CT Conversion Reagent - H2O 9.0mL - M-Dissolving Buffer 500ul - M-Dilution Buffer 3.0mL Mix at room temperature with frequent vortexing or shaking for 15 minutes Add 144 ml of 100% ethanol to the 36 ml M-Wash Buffer concentrate before use {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Lab IDs''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Volume for 1200ng''' | align="center" style="background:#f0f0f0;"|'''LabID ID''' | align="center" style="background:#f0f0f0;"|'''Well IDs''' | align="center" style="background:#f0f0f0;"|'''H2O''' | align="center" style="background:#f0f0f0;"|'''Total amount (ng)''' | align="center" style="background:#f0f0f0;"|'''CT conversion reagent (ul)''' | align="center" style="background:#f0f0f0;"|'''Total volume (ul)''' |- | Ap15-1||110.0||10.91||Ap15-1||A1||9.09||1200.00||130.00||150.00 |- | Ap15-2||139.1||8.63||Ap15-2||B1||11.37||1200.00||130.00||150.00 |- | Ap15-3||145.1||8.27||Ap15-3||C1||11.73||1200.00||130.00||150.00 |- | Ap15-4||295.5||4.06||Ap15-4||D1||15.94||1200.00||130.00||150.00 |- | Ap15-5||128.1||9.37||Ap15-5||E1||10.63||1200.00||130.00||150.00 |- | Ap15-6||153.9||7.80||Ap15-6||F1||12.20||1200.00||130.00||150.00 |- | Ap15-7||183.5||6.54||Ap15-7||G1||13.46||1200.00||130.00||150.00 |- | Ap15-8||149.3||8.04||Ap15-8||H1||11.96||1200.00||130.00||150.00 |- | Ap15-9||111.2||10.79||Ap15-9||A2||9.21||1200.00||130.00||150.00 |- | Ap15-10||153.5||7.82||Ap15-10||B2||12.18||1200.00||130.00||150.00 |- | Ap15-11||87.5||13.71||Ap15-11||C2||6.29||1200.00||130.00||150.00 |- | Ap15-12||109.7||10.94||Ap15-12||D2||9.06||1200.00||130.00||150.00 |- | Ap15-13||191.3||6.27||Ap15-13||E2||13.73||1200.00||130.00||150.00 |- | Ap15-14||108.7||11.04||Ap15-14||F2||8.96||1200.00||130.00||150.00 |- | Ap15-15||134.2||8.94||Ap15-15||G2||11.06||1200.00||130.00||150.00 |- | Ap15-16||50.2||20.00||Ap15-16||H2||0.00||1200.00||130.00||150.00 |- | Ap15-17||183.0||6.56||Ap15-17||A3||13.44||1200.00||130.00||150.00 |- | Ap15-18||82.6||14.53||Ap15-18||B3||5.47||1200.00||130.00||150.00 |- | Ap15-19||263.4||4.56||Ap15-19||C3||15.44||1200.00||130.00||150.00 |- | Ma15-1||86.7||13.84||Ma15-1||D3||6.16||1200.00||130.00||150.00 |- | Ma15-2||215.2||5.58||Ma15-2||E3||14.42||1200.00||130.00||150.00 |- | Ma15-3||252.0||4.76||Ma15-3||F3||15.24||1200.00||130.00||150.00 |- | Ma15-4||226.1||5.31||Ma15-4||G3||14.69||1200.00||130.00||150.00 |- | Ma15-5||238.7||5.03||Ma15-5||H3||14.97||1200.00||130.00||150.00 |- | Ma15-6||83.4||14.39||Ma15-6||A4||5.61||1200.00||130.00||150.00 |- | Ma15-7||236.8||5.07||Ma15-7||B4||14.93||1200.00||130.00||150.00 |- | Ma15-8||175.9||6.82||Ma15-8||C4||13.18||1200.00||130.00||150.00 |- | Ma15-9||164.3||7.30||Ma15-9||D4||12.70||1200.00||130.00||150.00 |- | Ma15-10||205.9||5.83||Ma15-10||E4||14.17||1200.00||130.00||150.00 |- | Ma15-11||97.6||12.30||Ma15-11||F4||7.70||1200.00||130.00||150.00 |- | Ma15-12||129.5||9.27||Ma15-12||G4||10.73||1200.00||130.00||150.00 |- | Ma15-13||263.1||4.56||Ma15-13||H4||15.44||1200.00||130.00||150.00 |- | Ma15-14||76.1||15.77||Ma15-14||A5||4.23||1200.00||130.00||150.00 |- | Ma15-15||138.7||8.65||Ma15-15||B5||11.35||1200.00||130.00||150.00 |- | Ma15-16||216.4||5.55||Ma15-16||C5||14.45||1200.00||130.00||150.00 |- | Ma15-17||137.6||8.72||Ma15-17||D5||11.28||1200.00||130.00||150.00 |- | Ma15-18||224.6||5.34||Ma15-18||E5||14.66||1200.00||130.00||150.00 |- | Ma15-19||111.7||10.74||Ma15-19||F5||9.26||1200.00||130.00||150.00 |- | Ma15-20||289.9||4.14||Ma15-20||G5||15.86||1200.00||130.00||150.00 |- | Ma15-21||172.9||6.94||Ma15-21||H5||13.06||1200.00||130.00||150.00 |} {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Lab IDs''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Volume for 1200ng''' | align="center" style="background:#f0f0f0;"|'''LabID ID''' | align="center" style="background:#f0f0f0;"|'''Well IDs''' | align="center" style="background:#f0f0f0;"|'''H2O''' | align="center" style="background:#f0f0f0;"|'''Total amount (ng)''' | align="center" style="background:#f0f0f0;"|'''CT conversion reagent (ul)''' | align="center" style="background:#f0f0f0;"|'''Total volume (ul)''' |- | SR-2||90.8||13.22||SR-2||B6||6.78||1200.00||130.00||150.00 |- | SR-10||184.8||6.49||SR-10||B7||13.51||1200.00||130.00||150.00 |- | SR-11||105.4||5.00||SR-11||C7||15.00||527.00||130.00||150.00 |- | SR-12||181.4||6.62||SR-12||D7||13.38||1200.00||130.00||150.00 |- | SR-13||270.0||4.44||SR-13||E7||15.56||1200.00||130.00||150.00 |- | SR-14||33.0||20.00||SR-14||F7||0.00||495.00||130.00||150.00 |- | SR-15||67.0||17.91||SR-15||G7||2.09||1200.00||130.00||150.00 |- | SR-16||145.6||8.24||SR-16||H7||11.76||1200.00||130.00||150.00 |- | SR-17||245.3||4.89||SR-17||A8||15.11||1200.00||130.00||150.00 |- | SR-19||159.5||7.52||SR-19||C8||12.48||1200.00||130.00||150.00 |- | SR-20||195.7||6.13||SR-20||D8||13.87||1200.00||130.00||150.00 |} {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Well IDs/Sample IDs''' | align="center" style="background:#f0f0f0;"|'''Well IDs/Sample IDs''' | align="center" style="background:#f0f0f0;"|'''Well IDs/Sample IDs''' | align="center" style="background:#f0f0f0;"|'''Well IDs/Sample IDs''' | align="center" style="background:#f0f0f0;"|'''Well IDs/Sample IDs''' | align="center" style="background:#f0f0f0;"|'''Well IDs/Sample IDs''' | align="center" style="background:#f0f0f0;"|'''Well IDs/Sample IDs''' | align="center" style="background:#f0f0f0;"|'''Well IDs/Sample IDs''' |- | A1||A2||A3||A4||A5||A6||A7||A8 |- | Ap15-1||Ap15-9||Ap15-17||Ma15-6||Ma15-14||SR-1 => NO||SR-9||SR-17 |- | B1||B2||B3||B4||B5||B6||B7||B8 |- | Ap15-2||Ap15-10||Ap15-18||Ma15-7||Ma15-15||SR-2||SR-10||SR-18 => NO |- | C1||C2||C3||C4||C5||C6||C7||C8 |- | Ap15-3||Ap15-11||Ap15-19||Ma15-8||Ma15-16||SR-3 => NO||SR-11||SR-19 |- | D1||D2||D3||D4||D5||D6||D7||D8 |- | Ap15-4||Ap15-12||Ma15-1||Ma15-9||Ma15-17||SR-4 => NO||SR-12||SR-20 |- | E1||E2||E3||E4||E5||E6||E7||E8 |- | Ap15-5||Ap15-13||Ma15-2||Ma15-10||Ma15-18||SR-5 => NO||SR-13||SR-21 => NO |- | F1||F2||F3||F4||F5||F6||F7||F8 |- | Ap15-6||Ap15-14||Ma15-3||Ma15-11||Ma15-19||SR-6 => NO||SR-14||SR-22 => NO |- | G1||G2||G3||G4||G5||G6||G7||G8 |- | Ap15-7||Ap15-15||Ma15-4||Ma15-12||Ma15-20||SR-7 => NO||SR-15|| |- | H1||H2||H3||H4||H5||H6||H7||H8 |- | Ap15-8||Ap15-16||Ma15-5||Ma15-13||Ma15-21||SR-8 => NO||SR-16|| |} Note: no more gDNA for SR-14 samples '''Program''' 98°C for 10 minutes 64°C for 2.5 hours 4°C storage up to 20 hours Measure conc. with ssDNA Qubit assay (used 1ul of bis-cvt gDNA) == Qubit ssDNA assay == * I combined bis-cvt gDNA of two batch together and measure concentration. * Sample in assay 1ul, dilution 200x {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Sample''' | align="center" style="background:#f0f0f0;"|'''Concentration in the Qubit''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''Final conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Yields in 30ul (ng)''' |- | A1||50.6||ng/mL||10.12||303.60 |- | B1||47.9||ng/mL||9.58||287.40 |- | C1||56.9||ng/mL||11.38||341.40 |- | D1||63.2||ng/mL||12.64||379.20 |- | E1||47.1||ng/mL||9.42||282.60 |- | F1||66.2||ng/mL||13.24||397.20 |- | G1||67.1||ng/mL||13.42||402.60 |- | H1||59.7||ng/mL||11.94||358.20 |- | A2||69||ng/mL||13.80||414.00 |- | B2||53.2||ng/mL||10.64||319.20 |- | C2||52.8||ng/mL||10.56||316.80 |- | D2||55.7||ng/mL||11.14||334.20 |- | E2||58.5||ng/mL||11.70||351.00 |- | F2||60.9||ng/mL||12.18||365.40 |- | G2||51.2||ng/mL||10.24||307.20 |- | H2||36.8||ng/mL||7.36||220.80 |- | A3||71.5||ng/mL||14.30||429.00 |- | B3||57.5||ng/mL||11.50||345.00 |- | C3||79.4||ng/mL||15.88||476.40 |- | D3||44.2||ng/mL||8.84||265.20 |- | E3||43||ng/mL||8.60||258.00 |- | F3||58.1||ng/mL||11.62||348.60 |- | G3||49.4||ng/mL||9.88||296.40 |- | H3||65.3||ng/mL||13.06||391.80 |- | A4||46.4||ng/mL||9.28||278.40 |- | B4||63.6||ng/mL||12.72||381.60 |- | C4||54.6||ng/mL||10.92||327.60 |- | D4||48.5||ng/mL||9.70||291.00 |- | E4||88.1||ng/mL||17.62||528.60 |- | F4||51.4||ng/mL||10.28||308.40 |- | G4||35.4||ng/mL||7.08||212.40 |- | H4||66.3||ng/mL||13.26||397.80 |- | A5||24.4||ng/mL||4.88||146.40 |- | B5||24.3||ng/mL||4.86||145.80 |- | C5||57.3||ng/mL||11.46||343.80 |- | D5||59.2||ng/mL||11.84||355.20 |- | E5||104||ng/mL||20.80||624.00 |- | F5||55.8||ng/mL||11.16||334.80 |- | G5||56.7||ng/mL||11.34||340.20 |- | H5||73.2||ng/mL||14.64||439.20 |- | B6||58||ng/mL||11.60||348.00 |- | B7||55.8||ng/mL||11.16||334.80 |- | D7||89.4||ng/mL||17.88||536.40 |- | E7||30.7||ng/mL||6.14||184.20 |- | G7||71||ng/mL||14.20||426.00 |- | H7||26.8||ng/mL||5.36||160.80 |- | A8||47.1||ng/mL||9.42||282.60 |- | C8||36.4||ng/mL||7.28||218.40 |- | D8||46.1||ng/mL||9.22||276.60 |}
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