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* [[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes '''Link to calendar''']] = DMR 330k BSPP capture of samples from Sergio = * Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-5-17 == Sample list == == New gDNA extracted from the cell pellet == on: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-5-17 {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''No.''' | align="center" style="background:#f0f0f0;"|'''Sample ID''' | align="center" style="background:#f0f0f0;"|'''Lab ID''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="background:#f0f0f0;"|'''Yields (ug)''' |- | 1||HUES2 p41||Ap15-1||110.0||250||27.5 |- | 2||ASThiPS4F1 p12||Ap15-2||139.1||250||34.8 |- | 3||ASThiPS4F2 p12||Ap15-3||145.1||250||36.3 |- | 4||HUES6 p39 Control TeSR||Ap15-4||295.5||250||73.9 |- | 5||HUES6 p39 ACTIVIN||Ap15-5||128.1||250||32.0 |- | 6||HUES6 p39 BMP-4||Ap15-6||153.9||250||38.5 |- | 7||HUES9 p39 Control TeSR||Ap15-7||183.5||250||45.9 |- | 8||HUES9 p39 ACTIVIN||Ap15-8||149.3||250||37.3 |- | 9||HUES9 p39 BMP-4||Ap15-9||111.2||250||27.8 |- | 10||HUES3 p36 Control TeSR||Ap15-10||153.5||250||38.4 |- | 11||HUES3 p36 ACTIVIN||Ap15-11||87.5||250||21.9 |- | 12||HUES3 p36 BMP-4||Ap15-12||109.7||250||27.4 |- | 13||H1 p54 Control TeSR||Ap15-13||191.3||250||47.8 |- | 14||H1 p54 ACTIVIN||Ap15-14||108.7||250||27.2 |- | 15||ASThiPS4F5 p16 Control TeSR||Ap15-15||134.2||250||33.6 |- | *16||ASThiPS4F5 p16 ACTIVIN ||Ap15-16||50.2||500||25.1 |- | 17||ASThiPS4F4 p36 Control TeSR||Ap15-17||183.0||250||45.8 |- | 18||ASThiPS4F4 p36 ACTIVIN||Ap15-18||82.6||250||20.7 |- | 19||ASThiPS4F4 p36 BMP-4||Ap15-19||263.4||250||65.9 |- | 20||H1 p48||Ma15-2||215.2||200||43.0 |- | 21||H9 p47 ||Ma15-3||252.0||200||50.4 |- | 22||HUES3 p33||Ma15-4||226.1||200||45.2 |- | 23||HUES6 p26||Ma15-5||238.7||200||47.7 |- | 24||HUES8 p35||Ma15-6||83.4||200||16.7 |- | 25||HUES9 p36||Ma15-7||236.8||200||47.4 |- | 26||ASThiPS4F3 p24 (derived from Astrocytes)||Ma15-8||175.9||200||35.2 |- | 27||ASThiPS4F4 p16 (derived from Astrocytes)||Ma15-9||164.3||200||32.9 |- | 28||FiPS4F2 p32 (derived from IMR90 fibroblasts)||Ma15-10||205.9||200||41.2 |- | 29||FiPS4F5 p29 (derived from IMR90 fibroblasts)||Ma15-11||97.6||200||19.5 |- | 30||KiPS4FA p54 (derived from keratinocytes)||Ma15-12||129.5||200||25.9 |- | 31||Huv-iPS4F6 p23 (derived from HUVECs)||Ma15-13||263.1||200||52.6 |- | 32||IMR90 p6||Ma15-14||76.1||200||15.2 |- | 33||Keratinocytes p3||Ma15-15||138.7||200||27.7 |- | 34||H1 p47 Control||Ma15-16||216.4||200||43.3 |- | 35||H1 p47 BMP-4||Ma15-17||137.6||200||27.5 |- | 36||ASThiPS4F5 p12 Control||Ma15-18||224.6||200||44.9 |- | 37||ASThiPS4F5 p12 BMP-4||Ma15-19||111.7||200||22.3 |- | 38||Huv-iPS4F6 p23 Control||Ma15-20||289.9||200||58.0 |- | 39||Huv-iPS4F6 p23 BMP-4||Ma15-21||172.9||200||34.6 |- | 40||Huv-iPS4F6 p23 ACTIVIN||Ma15-1||86.7||250||21.7 |} == Old gDNA of samples have been captured in the previous experiments == * Note: ** Some of gDNA concentrations were re-measured and note that some of them have conc. lower than 50ng/ul, and some of them don't have enough amount for bisulfite conversion ** Samples KERAT MMTA and KiPS4F #8 have high conc., but very little volume. I added ~17ul H2O then re-measured conc. again ** ***I may not do bis-cvt on the two samples, SR-21 and SR22 since the amount of gDNA was too little {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''No''' | align="center" style="background:#f0f0f0;"|'''Sample IDs''' | align="center" style="background:#f0f0f0;"|'''Lab IDs''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' |- | 1||H9 p43 mTeSR||SR-1||62.8 |- | 2||H9 p43 ACTIVIN ||SR-2||90.8 |- | 3||H9 p43 BMP4||SR-3||93.7 |- | 4||HUV0iPS4F1 p19||SR-4||98.0 |- | 5||HUV-hiPS4F1 p20 ACTIVIN||SR-5||84.8 |- | 6||HUV-hiPS4F1 p20 BMP4||SR-6||78.6 |- | 7||HUV-iPS4F3 F19||SR-7||96.9 |- | 8||HUV-hiPS4F3 p20 ACTIVIN||SR-8||91.9 |- | 9||HUV-hiPS4F3 p20 BMP4||SR-9||80.6 |- | 10||aMSCs p7||SR-10||184.8 |- | 11||MSCiPS #8 p14 (~4 ul)||SR-11||105.4 |- | 12||MSCiPS #4 p14||SR-12||181.4 |- | 13||hNiPS2F p23||SR-13||270.0 |- | 14||hKiPS3F-7 p24 (~15ul)||SR-14||33.0 |- | 15||hNSC ||SR-15||67.0 |- | 16||hFiPS4F-7 p9||SR-16||145.6 |- | 17||HUVEC p2||SR-17||245.3 |- | 18||hiPS AST4F-4 p9||SR-18||89.9 |- | 19||KERAT MMTA ||SR-19||159.5 |- | 20||KiPS4F #8||SR-20||195.7 |- | 21||***olf MSC 19.1ng/ul volume only 3-5ul||SR-21||19.1 |- | 22||***KiPS 14ng/ul volume only 3-5ul||SR-22||14.0 |} == 8 samples of bis-cvt gDNA from Dinh == * The list will be filled later and need to re-measure of the conc. with ssDNA Qubit assay = 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-1||62.8||19.11||SR-1||A6||0.89||1200.00||130.00||150.00 |- | SR-2||90.8||13.22||SR-2||B6||6.78||1200.00||130.00||150.00 |- | SR-3||93.7||12.81||SR-3||C6||7.19||1200.00||130.00||150.00 |- | SR-4||98.0||12.24||SR-4||D6||7.76||1200.00||130.00||150.00 |- | SR-5||84.8||14.15||SR-5||E6||5.85||1200.00||130.00||150.00 |- | SR-6||78.6||15.27||SR-6||F6||4.73||1200.00||130.00||150.00 |- | SR-7||96.9||12.38||SR-7||G6||7.62||1200.00||130.00||150.00 |- | SR-8||91.9||13.06||SR-8||H6||6.94||1200.00||130.00||150.00 |- | SR-9||80.6||14.89||SR-9||A7||5.11||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-18||89.9||13.35||SR-18||B8||6.65||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 |- | SR-21||19.1||5.00||SR-21||E8||15.00||95.50||130.00||150.00 |- | SR-22||14.0||5.00||SR-22||F8||15.00||70.00||130.00||150.00 |} '''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 == * Sample in assay 1ul, dilution 200x {| {{table}} border =1 | align="center" style="background:#f0f0f0;"|'''Sample''' | align="center" style="background:#f0f0f0;"|'''LabID ID''' | align="center" style="background:#f0f0f0;"|'''Well IDs''' | 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 16ul (ng)''' | align="center" style="background:#f0f0f0;"|'''LabID ID''' | align="center" style="background:#f0f0f0;"|'''Well IDs''' | align="center" style="background:#f0f0f0;"|'''Repeated samples''' |- | 1.00||Ap15-1||A1||57.00||ng/mL||11.40||182.40||Ap15-1||A1||X |- | 2.00||Ap15-2||B1||62.20||ng/mL||12.44||199.04||Ap15-2||B1||X |- | 3.00||Ap15-3||C1||81.70||ng/mL||16.34||261.44||Ap15-3||C1||X |- | 4.00||Ap15-4||D1||75.40||ng/mL||15.08||241.28||Ap15-4||D1||X |- | 5.00||Ap15-5||E1||59.60||ng/mL||11.92||190.72||Ap15-5||E1||X |- | 6.00||Ap15-6||F1||74.50||ng/mL||14.90||238.40||Ap15-6||F1||X |- | 7.00||Ap15-7||G1||76.40||ng/mL||15.28||244.48||Ap15-7||G1||X |- | 8.00||Ap15-8||H1||76.40||ng/mL||15.28||244.48||Ap15-8||H1||X |- | 9.00||Ap15-9||A2||76.10||ng/mL||15.22||243.52||Ap15-9||A2||X |- | 10.00||Ap15-10||B2||60.10||ng/mL||12.02||192.32||Ap15-10||B2||X |- | 11.00||Ap15-11||C2||56.40||ng/mL||11.28||180.48||Ap15-11||C2||X |- | 12.00||Ap15-12||D2||59.00||ng/mL||11.80||188.80||Ap15-12||D2||X |- | 13.00||Ap15-13||E2||72.50||ng/mL||14.50||232.00||Ap15-13||E2||X |- | 14.00||Ap15-14||F2||74.30||ng/mL||14.86||237.76||Ap15-14||F2||X |- | 15.00||Ap15-15||G2||58.90||ng/mL||11.78||188.48||Ap15-15||G2||X |- | 16.00||Ap15-16||H2||45.10||ng/mL||9.02||144.32||Ap15-16||H2||X |- | 17.00||Ap15-17||A3||76.40||ng/mL||15.28||244.48||Ap15-17||A3||X |- | 18.00||Ap15-18||B3||54.90||ng/mL||10.98||175.68||Ap15-18||B3||X |- | 19.00||Ap15-19||C3||78.90||ng/mL||15.78||252.48||Ap15-19||C3||X |- | 20.00||Ma15-1||D3||44.10||ng/mL||8.82||141.12||Ma15-1||D3||X |- | 21.00||Ma15-2||E3||49.80||ng/mL||9.96||159.36||Ma15-2||E3||X |- | 22.00||Ma15-3||F3||51.60||ng/mL||10.32||165.12||Ma15-3||F3||X |- | 23.00||Ma15-4||G3||58.20||ng/mL||11.64||186.24||Ma15-4||G3||X |- | 24.00||Ma15-5||H3||60.10||ng/mL||12.02||192.32||Ma15-5||H3||X |- | 25.00||Ma15-6||A4||40.60||ng/mL||8.12||129.92||Ma15-6||A4||X |- | 26.00||Ma15-7||B4||59.90||ng/mL||11.98||191.68||Ma15-7||B4||X |- | 27.00||Ma15-8||C4||54.50||ng/mL||10.90||174.40||Ma15-8||C4||X |- | 28.00||Ma15-9||D4||43.60||ng/mL||8.72||139.52||Ma15-9||D4||X |- | 29.00||Ma15-10||E4||73.10||ng/mL||14.62||233.92||Ma15-10||E4||X |- | 30.00||Ma15-11||F4||49.70||ng/mL||9.94||159.04||Ma15-11||F4||X |- | 31.00||Ma15-12||G4||36.90||ng/mL||7.38||118.08||Ma15-12||G4||X |- | 32.00||Ma15-13||H4||60.00||ng/mL||12.00||192.00||Ma15-13||H4||X |- | 33.00||Ma15-14||A5||28.00||ng/mL||5.60||89.60||Ma15-14||A5||X |- | 34.00||Ma15-15||B5||27.70||ng/mL||5.54||88.64||Ma15-15||B5||X |- | 35.00||Ma15-16||C5||53.50||ng/mL||10.70||171.20||Ma15-16||C5||X |- | 36.00||Ma15-17||D5||51.10||ng/mL||10.22||163.52||Ma15-17||D5||X |- | 37.00||Ma15-18||E5||53.60||ng/mL||10.72||171.52||Ma15-18||E5||X |- | 38.00||Ma15-19||F5||51.40||ng/mL||10.28||164.48||Ma15-19||F5||X |- | 39.00||Ma15-20||G5||48.90||ng/mL||9.78||156.48||Ma15-20||G5||X |- | 40.00||Ma15-21||H5||55.40||ng/mL||11.08||177.28||Ma15-21||H5||X |- | 1.00||H9 p43 mTeSR||SR-1||108.00||ng/mL||21.60||345.60||SR-1||A6||- |- | 2.00||H9 p43 ACTIVIN ||SR-2||64.90||ng/mL||12.98||207.68||SR-2||B6||X |- | 3.00||H9 p43 BMP4||SR-3||133.00||ng/mL||26.60||425.60||SR-3||C6||- |- | 4.00||HUV0iPS4F1 p19||SR-4||140.00||ng/mL||28.00||448.00||SR-4||D6||- |- | 5.00||HUV-hiPS4F1 p20 ACTIVIN||SR-5||95.40||ng/mL||19.08||305.28||SR-5||E6||- |- | 6.00||HUV-hiPS4F1 p20 BMP4||SR-6||135.00||ng/mL||27.00||432.00||SR-6||F6||- |- | 7.00||HUV-iPS4F3 F19||SR-7||134.00||ng/mL||26.80||428.80||SR-7||G6||- |- | 8.00||HUV-hiPS4F3 p20 ACTIVIN||SR-8||96.10||ng/mL||19.22||307.52||SR-8||H6||- |- | 9.00||HUV-hiPS4F3 p20 BMP4||SR-9||120.00||ng/mL||24.00||384.00||SR-9||A7||- |- | 10.00||aMSCs p7||SR-10||53.90||ng/mL||10.78||172.48||SR-10||B7||X |- | 11.00||MSCiPS #8 p14 (~4 ul)||SR-11||71.60||ng/mL||14.32||229.12||SR-11||C7||Can\'t repeat |- | 12.00||MSCiPS #4 p14||SR-12||94.60||ng/mL||18.92||302.72||SR-12||D7||- |- | 13.00||hNiPS2F p23||SR-13||29.60||ng/mL||5.92||94.72||SR-13||E7||X |- | 14.00||hKiPS3F-7 p24 (~15ul)||SR-14||7.13||ng/mL||1.43||22.82||SR-14||F7||X |- | 15.00||hNSC ||SR-15||70.90||ng/mL||14.18||226.88||SR-15||G7||X |- | 16.00||hFiPS4F-7 p9||SR-16||23.10||ng/mL||4.62||73.92||SR-16||H7||X |- | 17.00||HUVEC p2||SR-17||50.80||ng/mL||10.16||162.56||SR-17||A8||X |- | 18.00||hiPS AST4F-4 p9||SR-18||131.00||ng/mL||26.20||419.20||SR-18||B8|| |- | 19.00||KERAT MMTA ||SR-19||19.80||ng/mL||3.96||63.36||SR-19||C8||X |- | 20.00||KiPS4F #8||SR-20||19.00||ng/mL||3.80||60.80||SR-20||D8||X |- | 21.00||olf MSC 19.1ng/ul volume only 3-5ul||SR-21||4.60||ng/mL||0.92||14.72||SR-21||E8||can\'t repeat |- | 22.00||KiPS 14ng/ul volume only 3-5ul||SR-22||7.42||ng/mL||1.48||23.74||SR-22||F8||can\'t repeat |} * From the yields of bis-cvt gDNA, many samples have the yields lower than 300ng or 200ng which is the amount the acceptable for capture with BSPP. I checked the yields of the old samples I have processed before (SR-1 - SR-9) to see if this is the technical error of this batch. I got the total yields of SR-1 -15 pretty much the same as I got before. This suggested that there are not the batch effect, but may be the quality of gDNA. I need to repeat bis-cvt again to get enough yield for capture. However, there are some sample that has no more gDNA or has very low concentration, and I don't expected to get higher yield from those samples. * Continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-5-21
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