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= Repeat BSPP DMR330k capture of GA and control gDNA from blood sample = * 2011_08_08: received new plate of GA sample plate1 (so called GA plate1-2) from Ling. She prepared gDNA concentration 100ng/ul and volume ~10-15ul) * However, after measured the conc. by Nanodrop the conc. of the samples were very varied and were not enough for bisulfite conversion. The concentrations in plate 1-1 were verified by Nanodrop again. Then I calculated the volume required for 1200ng. I didn't pool the gDNA from the two plates because they have different conc. and some sample has too low conc. = Bisulfite conversion = * Used EZ-96 DNA Methylation-Goldβ’ Kit (deep well format) Cat#D5008 * Eluted volume 20ul with Elution buffer. After Qubit ss-DNA assay the total yields will be calculated based on ~16ul left in the well. {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Sample ID''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Volume plate1-2 enough?''' | align="center" style="background:#f0f0f0;"|'''Volume plate1-2 (ul)''' | align="center" style="background:#f0f0f0;"|'''Amount plate1-2''' | align="center" style="background:#f0f0f0;"|'''Conc. plate#1-1 (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Volume plate 1-1''' | align="center" style="background:#f0f0f0;"|'''Sample ID''' | align="center" style="background:#f0f0f0;"|'''H2O (ul)''' | align="center" style="background:#f0f0f0;"|'''Total volume (ul)''' | align="center" style="background:#f0f0f0;"|'''CT conversion reagent (ul)''' | align="center" style="background:#f0f0f0;"|'''Total volume (ul)''' |- | A1||177.30||||6.77||1200.00||||||A1||13.23||20.00||130.00||150.00 |- | B1||104.91||||11.44||1200.00||||||B1||8.56||20.00||130.00||150.00 |- | C1||108.20||||11.09||1200.00||||||C1||8.91||20.00||130.00||150.00 |- | D1||95.30||||12.59||1200.00||||||D1||7.41||20.00||130.00||150.00 |- | E1||142.71||||8.41||1200.00||||||E1||11.59||20.00||130.00||150.00 |- | F1||130.28||||9.21||1200.00||||||F1||10.79||20.00||130.00||150.00 |- | G1||195.56||||6.14||1200.00||||||G1||13.86||20.00||130.00||150.00 |- | H1||126.34||||9.50||1200.00||||||H1||10.50||20.00||130.00||150.00 |- | A2||119.33||||10.06||1200.00||||||A2||9.94||20.00||130.00||150.00 |- | B2||135.56||||8.85||1200.00||||||B2||11.15||20.00||130.00||150.00 |- | C2||48.65||no||10.00||486.50||94.48||7.55||C2||2.45||20.00||130.00||150.00 |- | D2||242.84||||4.94||1200.00||||||D2||15.06||20.00||130.00||150.00 |- | E2||109.93||||10.92||1200.00||||||E2||9.08||20.00||130.00||150.00 |- | F2||139.81||||8.58||1200.00||||||F2||11.42||20.00||130.00||150.00 |- | G2||87.91||||13.65||1200.00||||||G2||6.35||20.00||130.00||150.00 |- | H2||64.74||no||15.00||971.10||138.55||1.65||H2||3.35||20.00||130.00||150.00 |- | A3||121.07||||9.91||1200.00||||||A3||10.09||20.00||130.00||150.00 |- | B3||103.64||||11.58||1200.00||||||B3||8.42||20.00||130.00||150.00 |- | C3||76.09||no||12.00||913.08||144.84||1.98||C3||6.02||20.00||130.00||150.00 |- | D3||154.51||||7.77||1200.00||||||D3||12.23||20.00||130.00||150.00 |- | E3||198.30||||6.05||1200.00||||||E3||13.95||20.00||130.00||150.00 |- | F3||138.66||||8.65||1200.00||||||F3||11.35||20.00||130.00||150.00 |- | G3||163.20||||7.35||1200.00||||||G3||12.65||20.00||130.00||150.00 |- | H3||56.20||no||10.00||562.00||124.85||5.11||H3||4.89||20.00||130.00||150.00 |- | A4||98.78||||12.15||1200.00||||||A4||7.85||20.00||130.00||150.00 |- | B4||60.47||no||12.00||725.64||151.88||3.12||B4||4.88||20.00||130.00||150.00 |- | C4||66.43||no||12.00||797.16||119.93||3.36||C4||4.64||20.00||130.00||150.00 |- | D4||119.64||||10.03||1200.00||||||D4||9.97||20.00||130.00||150.00 |- | E4||208.77||||5.75||1200.00||||||E4||14.25||20.00||130.00||150.00 |- | F4||83.48||no||12.00||1001.76||58.64||3.38||F4||4.62||20.00||130.00||150.00 |- | G4||71.01||no||12.00||852.12||36.48||9.54||G4||0.00||21.54||130.00||151.54 |- | H4||210.32||||5.71||1200.00||||||H4||14.29||20.00||130.00||150.00 |- | A5||266.74||||4.50||1200.00||||||A5||15.50||20.00||130.00||150.00 |- | B5||105.58||||11.37||1200.00||||||B5||8.63||20.00||130.00||150.00 |- | C5||89.41||||13.42||1200.00||||||C5||6.58||20.00||130.00||150.00 |- | D5||61.84||no||15.00||927.60||177.45||1.54||D5||3.46||20.00||130.00||150.00 |- | E5||66.71||no||12.00||800.52||135.97||2.94||E5||5.06||20.00||130.00||150.00 |- | F5||125.29||||9.58||1200.00||||||F5||10.42||20.00||130.00||150.00 |- | G5||121.31||||9.89||1200.00||||||G5||10.11||20.00||130.00||150.00 |- | H5||88.27||||13.59||1200.00||||||H5||6.41||20.00||130.00||150.00 |- | A6||134.70||||8.91||1200.00||||||A6||11.09||20.00||130.00||150.00 |- | B6||62.68||no||12.00||752.16||154.46||2.90||B6||5.10||20.00||130.00||150.00 |- | C6||141.72||||8.47||1200.00||||||C6||11.53||20.00||130.00||150.00 |- | D6||187.04||||6.42||1200.00||||||D6||13.58||20.00||130.00||150.00 |- | E6||64.02||no||12.00||768.24||138.92||3.11||E6||4.89||20.00||130.00||150.00 |- | F6||58.78||no ||12.00||705.36||188.15||2.63||F6||5.37||20.00||130.00||150.00 |- | G6||170.00||||7.06||1200.00||||||G6||12.94||20.00||130.00||150.00 |- | H6||111.00||||10.81||1200.00||||||H6||9.19||20.00||130.00||150.00 |- | A7||320.17||||3.75||1200.00||||||A7||16.25||20.00||130.00||150.00 |- | B7||162.36||||7.39||1200.00||||||B7||12.61||20.00||130.00||150.00 |- | C7||126.32||||9.50||1200.00||||||C7||10.50||20.00||130.00||150.00 |- | D7||368.67||||3.25||1200.00||||||D7||16.75||20.00||130.00||150.00 |- | E7||117.82||||10.19||1200.00||||||E7||9.81||20.00||130.00||150.00 |- | F7||84.38||no||12.00||1012.56||107.02||1.75||F7||6.25||20.00||130.00||150.00 |- | G7||105.04||||11.42||1200.00||||||G7||8.58||20.00||130.00||150.00 |- | H7||86.61||||13.86||1200.00||||||H7||6.14||20.00||130.00||150.00 |- | A8||95.35||||12.59||1200.00||||||A8||7.41||20.00||130.00||150.00 |- | B8||98.32||||12.21||1200.00||||||B8||7.79||20.00||130.00||150.00 |- | C8||129.29||||9.28||1200.00||||||C8||10.72||20.00||130.00||150.00 |- | D8||100.98||||11.88||1200.00||||||D8||8.12||20.00||130.00||150.00 |- | E8||73.13||no||12.00||877.56||132.82||2.43||E8||5.57||20.00||130.00||150.00 |- | F8||111.10||||10.80||1200.00||||||F8||9.20||20.00||130.00||150.00 |- | G8||30.00||no||10.00||300.00||162.10||5.55||G8||4.45||20.00||130.00||150.00 |- | H8||82.39||no||12.00||988.68||182.77||1.16||H8||6.84||20.00||130.00||150.00 |- | A9||61.85||no||12.00||742.20||136.13||3.36||A9||4.64||20.00||130.00||150.00 |- | B9||110.49||||10.86||1200.00||||||B9||9.14||20.00||130.00||150.00 |- | C9||62.30||no||12.00||747.60||123.23||3.67||C9||4.33||20.00||130.00||150.00 |- | D9||118.40||||10.14||1200.00||||||D9||9.86||20.00||130.00||150.00 |- | E9||75.01||||16.00||1200.00||||||E9||4.00||20.00||130.00||150.00 |- | F9 (tube 49.9)||60.63||no||15.00||909.45||38.24||7.60||F9 (tube 49.9)||0.00||22.60||130.00||152.60 |- | G9||91.20||no||12.00||1094.40||142.40||0.74||G9||7.26||20.00||130.00||150.00 |- | H9||90.62||||13.24||1200.00||||||H9||6.76||20.00||130.00||150.00 |- | A10||83.92||no||13.00||1090.96||193.87||0.56||A10||6.44||20.00||130.00||150.00 |- | B10||120.56||||9.95||1200.00||||||B10||10.05||20.00||130.00||150.00 |- | C10||86.38||no||12.00||1036.56||168.48||0.97||C10||7.03||20.00||130.00||150.00 |- | D10||49.85||no||12.00||598.20||172.60||3.49||D10||4.51||20.00||130.00||150.00 |- | E10||123.63||||9.71||1200.00||||||E10||10.29||20.00||130.00||150.00 |- | F10||14.38||no||12.00||172.56||89.90||11.43||F10||0.00||23.43||130.00||153.43 |- | G10||60.43||no||12.00||725.16||163.82||2.90||G10||5.10||20.00||130.00||150.00 |- | H10||71.01||no ||12.00||852.12||251.70||1.38||H10||6.62||20.00||130.00||150.00 |- | A11||71.94||no||12.00||863.28||96.54||3.49||A11||4.51||20.00||130.00||150.00 |- | B11||72.80||no||12.00||873.60||181.22||1.80||B11||6.20||20.00||130.00||150.00 |- | C11||43.85||no||12.00||526.20||192.17||3.51||C11||4.49||20.00||130.00||150.00 |- | D11||201.88||||5.94||1200.00||||||D11||14.06||20.00||130.00||150.00 |- | E11||221.63||||5.41||1200.00||||||E11||14.59||20.00||130.00||150.00 |- | F11||68.94||no||12.00||827.28||139.05||2.68||F11||5.32||20.00||130.00||150.00 |- | G11||76.28||no||12.00||915.36||137.85||2.06||G11||5.94||20.00||130.00||150.00 |- | H11||73.95||no||12.00||887.40||126.45||2.47||H11||5.53||20.00||130.00||150.00 |- | A12||88.25||no||12.00||1059.00||126.33||1.12||A12||6.88||20.00||130.00||150.00 |- | B12 (tube 94.9)||109.45||||10.96||1200.00||||||B12 (tube 94.9)||9.04||20.00||130.00||150.00 |- | C12||59.30||no||12.00||711.60||147.66||3.31||C12||4.69||20.00||130.00||150.00 |- | D12||42.39||no||12.00||508.68||153.07||4.52||D12||3.48||20.00||130.00||150.00 |- | E12||63.68||no||12.00||764.16||138.06||3.16||E12||4.84||20.00||130.00||150.00 |- | F12||26.25||no||12.00||315.00||144.07||6.14||F12||1.86||20.00||130.00||150.00 |- | G12||59.01||no||12.00||708.12||123.67||3.98||G12||4.02||20.00||130.00||150.00 |- | H12||142.64||||8.41||1200.00||||||H12||11.59||20.00||130.00||150.00 |}<br> = Qubit ssDNA assay = * Follow protocol provided with the kit * Took 1ul of bis-cvt DNA into 9ul of H2O, then add the diluted DNA to 190ul ssDN Qubit reagent (dilution factor = 200) {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Order''' | align="center" style="background:#f0f0f0;"|'''Well ID''' | align="center" style="background:#f0f0f0;"|'''Conc. in Qubit (ng/mL)''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="background:#f0f0f0;"|'''Final conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Yields (ng)''' |- | 1.00||A1||217.00||16.00||43.40||694.40 |- | 2.00||B1||180.00||16.00||36.00||576.00 |- | 3.00||C1||204.00||16.00||40.80||652.80 |- | 4.00||D1||211.00||16.00||42.20||675.20 |- | 5.00||E1||201.00||16.00||40.20||643.20 |- | 6.00||F1||150.00||16.00||30.00||480.00 |- | 7.00||G1||135.00||16.00||27.00||432.00 |- | 8.00||H1||219.00||16.00||43.80||700.80 |- | 9.00||A2||190.00||16.00||38.00||608.00 |- | 10.00||B2||170.00||16.00||34.00||544.00 |- | 11.00||C2||151.00||16.00||30.20||483.20 |- | 12.00||D2||153.00||16.00||30.60||489.60 |- | 13.00||E2||292.00||16.00||58.40||934.40 |- | 14.00||F2||112.00||16.00||22.40||358.40 |- | 15.00||G2||160.00||16.00||32.00||512.00 |- | 16.00||H2||105.00||16.00||21.00||336.00 |- | 17.00||A3||176.00||16.00||35.20||563.20 |- | 18.00||B3||137.00||16.00||27.40||438.40 |- | 19.00||C3||119.00||16.00||23.80||380.80 |- | 20.00||D3||128.00||16.00||25.60||409.60 |- | 21.00||E3*||40.20||16.00||8.04||128.64 |- | 22.00||F3||134.00||16.00||26.80||428.80 |- | 23.00||G3||176.00||16.00||35.20||563.20 |- | 24.00||H3||112.00||16.00||22.40||358.40 |- | 25.00||A4||144.00||16.00||28.80||460.80 |- | 26.00||B4||135.00||16.00||27.00||432.00 |- | 27.00||C4||133.00||16.00||26.60||425.60 |- | 28.00||D4||139.00||16.00||27.80||444.80 |- | 29.00||E4||133.00||16.00||26.60||425.60 |- | 30.00||F4||189.00||16.00||37.80||604.80 |- | 31.00||G4||88.60||16.00||17.72||283.52 |- | 32.00||H4*||47.10||16.00||9.42||150.72 |- | 33.00||A5||148.00||16.00||29.60||473.60 |- | 34.00||B5||127.00||16.00||25.40||406.40 |- | 35.00||C5||83.70||16.00||16.74||267.84 |- | 36.00||D5||120.00||16.00||24.00||384.00 |- | 37.00||E5||117.00||16.00||23.40||374.40 |- | 38.00||F5*||34.40||16.00||6.88||110.08 |- | 39.00||G5||104.00||16.00||20.80||332.80 |- | 40.00||H5||112.00||16.00||22.40||358.40 |- | 41.00||A6||125.00||16.00||25.00||400.00 |- | 42.00||B6||126.00||16.00||25.20||403.20 |- | 43.00||C6||149.00||16.00||29.80||476.80 |- | 44.00||D6||135.00||16.00||27.00||432.00 |- | 45.00||E6||110.00||16.00||22.00||352.00 |- | 46.00||F6||102.00||16.00||20.40||326.40 |- | 47.00||G6||127.00||16.00||25.40||406.40 |- | 48.00||H6||59.40||16.00||11.88||190.08 |- | 49.00||A7||161.00||16.00||32.20||515.20 |- | 50.00||B7||146.00||16.00||29.20||467.20 |- | 51.00||C7||160.00||16.00||32.00||512.00 |- | 52.00||D7||135.00||16.00||27.00||432.00 |- | 53.00||E7||146.00||16.00||29.20||467.20 |- | 54.00||F7||133.00||16.00||26.60||425.60 |- | 55.00||G7||143.00||16.00||28.60||457.60 |- | 56.00||H7||85.20||16.00||17.04||272.64 |- | 57.00||A8||141.00||16.00||28.20||451.20 |- | 58.00||B8||147.00||16.00||29.40||470.40 |- | 59.00||C8||119.00||16.00||23.80||380.80 |- | 60.00||D8||81.40||16.00||16.28||260.48 |- | 61.00||E8||120.00||16.00||24.00||384.00 |- | 62.00||F8||62.20||16.00||12.44||199.04 |- | 63.00||G8||106.00||16.00||21.20||339.20 |- | 64.00||H8||135.00||16.00||27.00||432.00 |- | 65.00||A9||110.00||16.00||22.00||352.00 |- | 66.00||B9||147.00||16.00||29.40||470.40 |- | 67.00||C9||127.00||16.00||25.40||406.40 |- | 68.00||D9||95.70||16.00||19.14||306.24 |- | 69.00||E9||99.80||16.00||19.96||319.36 |- | 70.00||F9||104.00||16.00||20.80||332.80 |- | 71.00||G9||92.50||16.00||18.50||296.00 |- | 72.00||H9||116.00||16.00||23.20||371.20 |- | 73.00||A10||103.00||16.00||20.60||329.60 |- | 74.00||B10||69.70||16.00||13.94||223.04 |- | 75.00||C10||121.00||16.00||24.20||387.20 |- | 76.00||D10||141.00||16.00||28.20||451.20 |- | 77.00||E10||114.00||16.00||22.80||364.80 |- | 78.00||F10||154.00||16.00||30.80||492.80 |- | 79.00||G10||124.00||16.00||24.80||396.80 |- | 80.00||H10||111.00||16.00||22.20||355.20 |- | 81.00||A11||82.20||16.00||16.44||263.04 |- | 82.00||B11||112.00||16.00||22.40||358.40 |- | 83.00||C11||109.00||16.00||21.80||348.80 |- | 84.00||D11||61.50||16.00||12.30||196.80 |- | 85.00||E11||65.20||16.00||13.04||208.64 |- | 86.00||F11||148.00||16.00||29.60||473.60 |- | 87.00||G11||133.00||16.00||26.60||425.60 |- | 88.00||H11||200.00||16.00||40.00||640.00 |- | 89.00||A12||114.00||16.00||22.80||364.80 |- | 90.00||B12||149.00||16.00||29.80||476.80 |- | 91.00||C12||164.00||16.00||32.80||524.80 |- | 92.00||D12||142.00||16.00||28.40||454.40 |- | 93.00||E12||145.00||16.00||29.00||464.00 |- | 94.00||F12||137.00||16.00||27.40||438.40 |- | 95.00||G12||177.00||16.00||35.40||566.40 |- | 96.00||H12||202.00||16.00||40.40||646.40 |}<br> * Note: * = samples which total yields < 200ng = 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 105rxn (ul)''' |- | DMR.s1||0.05||2.24||0.11||1.86||0.06||6.32 |- | DMR.s2||0.07||2.24||0.16||1.95||0.08||8.77 |- | DMR.s3||0.23||18.90||4.41||23.20||0.19||19.94 |- | DMR.s4||1.00||18.90||18.90||23.20||0.81||85.54 |- | DMR.s5||4.83||18.90||91.29||31.20||2.93||307.22 |- | DMR.Exp1||0.23||18.90||4.44||31.88||0.14||14.61 |- | DMR.Exp2||0.19||18.90||3.50||24.11||0.15||15.26 |- | DMR.Exp3||0.27||18.90||5.03||28.90||0.17||18.26 |- | 1.05uM Oligo suppressor-set1||||||||||0.15||15.75 |- | 1.05uM Oligo suppressor-set2||||||||||0.15||15.75 |}<br> * check volume of every well if they have the same level == Reaction mix == {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1rxn (ul)''' | align="center" style="background:#f0f0f0;"|'''105 rxn mix (ul)''' |- | 10X AmpLigase Buffer||2.50||262.50 |- | DMR330k probe mix (ul)||4.53||475.65 |- | 105nM Oligo suppressor||0.30||31.50 |- | Total volume (ul)||7.33||769.65 |}<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 300ng(ul)''' | align="center" style="background:#f0f0f0;"|'''sample ID''' | 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;"|'''1.05uM Oligo suppressor set1&2 (ul)''' | align="center" style="background:#f0f0f0;"|'''Total volume (ul)''' |- | A1||43.40||6.91||A1||10.76||300.00||4.53||2.50||0.30||25.00 |- | B1||36.00||8.33||B1||9.34||300.00||4.53||2.50||0.30||25.00 |- | C1||40.80||7.35||C1||10.32||300.00||4.53||2.50||0.30||25.00 |- | D1||42.20||7.11||D1||10.56||300.00||4.53||2.50||0.30||25.00 |- | E1||40.20||7.46||E1||10.21||300.00||4.53||2.50||0.30||25.00 |- | F1||30.00||10.00||F1||7.67||300.00||4.53||2.50||0.30||25.00 |- | G1||27.00||11.11||G1||6.56||300.00||4.53||2.50||0.30||25.00 |- | H1||43.80||6.85||H1||10.82||300.00||4.53||2.50||0.30||25.00 |- | A2||38.00||7.89||A2||9.78||300.00||4.53||2.50||0.30||25.00 |- | B2||34.00||8.82||B2||8.85||300.00||4.53||2.50||0.30||25.00 |- | C2||30.20||9.93||C2||7.74||300.00||4.53||2.50||0.30||25.00 |- | D2||30.60||9.80||D2||7.87||300.00||4.53||2.50||0.30||25.00 |- | E2||58.40||5.14||E2||12.53||300.00||4.53||2.50||0.30||25.00 |- | F2||22.40||13.39||F2||4.28||300.00||4.53||2.50||0.30||25.00 |- | G2||32.00||9.38||G2||8.30||300.00||4.53||2.50||0.30||25.00 |- | H2||21.00||14.29||H2||3.38||300.00||4.53||2.50||0.30||25.00 |- | A3||35.20||8.52||A3||9.15||300.00||4.53||2.50||0.30||25.00 |- | B3||27.40||10.95||B3||6.72||300.00||4.53||2.50||0.30||25.00 |- | C3||23.80||12.61||C3||5.06||300.00||4.53||2.50||0.30||25.00 |- | D3||25.60||11.72||D3||5.95||300.00||4.53||2.50||0.30||25.00 |- | E3*||8.04||16.00||E3*||1.67||128.64||4.53||2.50||0.30||25.00 |- | F3||26.80||11.19||F3||6.48||300.00||4.53||2.50||0.30||25.00 |- | G3||35.20||8.52||G3||9.15||300.00||4.53||2.50||0.30||25.00 |- | H3||22.40||13.39||H3||4.28||300.00||4.53||2.50||0.30||25.00 |- | A4||28.80||10.42||A4||7.25||300.00||4.53||2.50||0.30||25.00 |- | B4||27.00||11.11||B4||6.56||300.00||4.53||2.50||0.30||25.00 |- | C4||26.60||11.28||C4||6.39||300.00||4.53||2.50||0.30||25.00 |- | D4||27.80||10.79||D4||6.88||300.00||4.53||2.50||0.30||25.00 |- | E4||26.60||11.28||E4||6.39||300.00||4.53||2.50||0.30||25.00 |- | F4||37.80||7.94||F4||9.73||300.00||4.53||2.50||0.30||25.00 |- | G4||17.72||16.00||G4||1.67||283.52||4.53||2.50||0.30||25.00 |- | H4*||9.42||16.00||H4*||1.67||150.72||4.53||2.50||0.30||25.00 |- | A5||29.60||10.14||A5||7.53||300.00||4.53||2.50||0.30||25.00 |- | B5||25.40||11.81||B5||5.86||300.00||4.53||2.50||0.30||25.00 |- | C5||16.74||16.00||C5||1.67||267.84||4.53||2.50||0.30||25.00 |- | D5||24.00||12.50||D5||5.17||300.00||4.53||2.50||0.30||25.00 |- | E5||23.40||12.82||E5||4.85||300.00||4.53||2.50||0.30||25.00 |- | F5*||6.88||16.00||F5*||1.67||110.08||4.53||2.50||0.30||25.00 |- | G5||20.80||14.42||G5||3.25||300.00||4.53||2.50||0.30||25.00 |- | H5||22.40||13.39||H5||4.28||300.00||4.53||2.50||0.30||25.00 |- | A6||25.00||12.00||A6||5.67||300.00||4.53||2.50||0.30||25.00 |- | B6||25.20||11.90||B6||5.77||300.00||4.53||2.50||0.30||25.00 |- | C6||29.80||10.07||C6||7.60||300.00||4.53||2.50||0.30||25.00 |- | D6||27.00||11.11||D6||6.56||300.00||4.53||2.50||0.30||25.00 |- | E6||22.00||13.64||E6||4.03||300.00||4.53||2.50||0.30||25.00 |- | F6||20.40||14.71||F6||2.96||300.00||4.53||2.50||0.30||25.00 |- | G6||25.40||11.81||G6||5.86||300.00||4.53||2.50||0.30||25.00 |- | H6||11.88||16.00||H6||1.67||190.08||4.53||2.50||0.30||25.00 |- | A7||32.20||9.32||A7||8.35||300.00||4.53||2.50||0.30||25.00 |- | B7||29.20||10.27||B7||7.40||300.00||4.53||2.50||0.30||25.00 |- | C7||32.00||9.38||C7||8.30||300.00||4.53||2.50||0.30||25.00 |- | D7||27.00||11.11||D7||6.56||300.00||4.53||2.50||0.30||25.00 |- | E7||29.20||10.27||E7||7.40||300.00||4.53||2.50||0.30||25.00 |- | F7||26.60||11.28||F7||6.39||300.00||4.53||2.50||0.30||25.00 |- | G7||28.60||10.49||G7||7.18||300.00||4.53||2.50||0.30||25.00 |- | H7||17.04||16.00||H7||1.67||272.64||4.53||2.50||0.30||25.00 |- | A8||28.20||10.64||A8||7.03||300.00||4.53||2.50||0.30||25.00 |- | B8||29.40||10.20||B8||7.47||300.00||4.53||2.50||0.30||25.00 |- | C8||23.80||12.61||C8||5.06||300.00||4.53||2.50||0.30||25.00 |- | D8||16.28||16.00||D8||1.67||260.48||4.53||2.50||0.30||25.00 |- | E8||24.00||12.50||E8||5.17||300.00||4.53||2.50||0.30||25.00 |- | F8||12.44||16.00||F8||1.67||199.04||4.53||2.50||0.30||25.00 |- | G8||21.20||14.15||G8||3.52||300.00||4.53||2.50||0.30||25.00 |- | H8||27.00||11.11||H8||6.56||300.00||4.53||2.50||0.30||25.00 |- | A9||22.00||13.64||A9||4.03||300.00||4.53||2.50||0.30||25.00 |- | B9||29.40||10.20||B9||7.47||300.00||4.53||2.50||0.30||25.00 |- | C9||25.40||11.81||C9||5.86||300.00||4.53||2.50||0.30||25.00 |- | D9||19.14||15.67||D9||2.00||300.00||4.53||2.50||0.30||25.00 |- | E9||19.96||15.03||E9||2.64||300.00||4.53||2.50||0.30||25.00 |- | F9||20.80||14.42||F9||3.25||300.00||4.53||2.50||0.30||25.00 |- | G9||18.50||16.00||G9||1.67||296.00||4.53||2.50||0.30||25.00 |- | H9||23.20||12.93||H9||4.74||300.00||4.53||2.50||0.30||25.00 |- | A10||20.60||14.56||A10||3.11||300.00||4.53||2.50||0.30||25.00 |- | B10||13.94||16.00||B10||1.67||223.04||4.53||2.50||0.30||25.00 |- | C10||24.20||12.40||C10||5.27||300.00||4.53||2.50||0.30||25.00 |- | D10||28.20||10.64||D10||7.03||300.00||4.53||2.50||0.30||25.00 |- | E10||22.80||13.16||E10||4.51||300.00||4.53||2.50||0.30||25.00 |- | F10||30.80||9.74||F10||7.93||300.00||4.53||2.50||0.30||25.00 |- | G10||24.80||12.10||G10||5.57||300.00||4.53||2.50||0.30||25.00 |- | H10||22.20||13.51||H10||4.16||300.00||4.53||2.50||0.30||25.00 |- | A11||16.44||16.00||A11||1.67||263.04||4.53||2.50||0.30||25.00 |- | B11||22.40||13.39||B11||4.28||300.00||4.53||2.50||0.30||25.00 |- | C11||21.80||13.76||C11||3.91||300.00||4.53||2.50||0.30||25.00 |- | D11||12.30||16.00||D11||1.67||196.80||4.53||2.50||0.30||25.00 |- | E11||13.04||16.00||E11||1.67||208.64||4.53||2.50||0.30||25.00 |- | F11||29.60||10.14||F11||7.53||300.00||4.53||2.50||0.30||25.00 |- | G11||26.60||11.28||G11||6.39||300.00||4.53||2.50||0.30||25.00 |- | H11||40.00||7.50||H11||10.17||300.00||4.53||2.50||0.30||25.00 |- | A12||22.80||13.16||A12||4.51||300.00||4.53||2.50||0.30||25.00 |- | B12||29.80||10.07||B12||7.60||300.00||4.53||2.50||0.30||25.00 |- | C12||32.80||9.15||C12||8.52||300.00||4.53||2.50||0.30||25.00 |- | D12||28.40||10.56||D12||7.11||300.00||4.53||2.50||0.30||25.00 |- | E12||29.00||10.34||E12||7.33||300.00||4.53||2.50||0.30||25.00 |- | F12||27.40||10.95||F12||6.72||300.00||4.53||2.50||0.30||25.00 |- | G12||35.40||8.47||G12||9.20||300.00||4.53||2.50||0.30||25.00 |- | H12||40.40||7.43||H12||10.24||300.00||4.53||2.50||0.30||25.00 |}<br> * Add H2O to the 96-well plate first then add bis-cvt gDNA. Note: Try to deliver reagents to the bottom of the well * Prepare reaction mix in 1.5 mL tube by pipetting probe, oligo suppressor and 10x AmLigase buffer and mix well. * Aliquot reaction mix in striptube (8 tubes): 96.21 ul * Aliquot reaction mix 7.33 ul with multichannel pipette and mix well * Put mineral oil in basin ~3 mL) * Layer the capture reaction with 20ul mineral oil with multichannel pipette. * Seal the plate with microseal and make sure that every well is completely sealed to prevent evaporation * Check the level of every well that they have the same level * Centrifuge the capture plate for 3 min and place the plate on PCR machine with the following profile, '''Program'''<br> -> 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_08_14) -> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold. (2011_08_15) == SLN mix == * Prepare 275 ul (2.5ul/rxn and prepare total for 110 rxn, more extra reaction to ensure that the volume enough when pipetting)<br> {| {{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;"|'''Prepared volume 275ul''' |- | AmpliTaq Stoffel||10.00||U/ul||2.00||U/ul||55.00 |- | AmpLigase||5.00||U/ul||0.50||U/ul||27.50 |- | dNTP||1000.00||uM||100.00||uM||27.50 |- | 10x AmpLigase Buffer||10.00||x||1.00||x||27.50 |- | H2O||||||||||137.50 |- | Total||||||||||275.00 |} * Aliquot 34.38 of SLN mix to strip tube. * Add 2.5ul to each well with multi-channel pipette. == ExoI/III preparation == * Mix 110 ul each of ExoI and ExoIII and aliquot 27.5ul into striptube * Add 2ul to each well with multichannel pipettes = PCR amplification of captured DNA = '''Plate1''' {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1rxn''' | align="center" style="background:#f0f0f0;"|'''100 rxn mix''' |- | Captured template||10.00||0.00 |- | 100uM AmpF6.4Sol||0.20||20.00 |- | 10uM AmpR6.3 (IndX, X 1,2,3,β¦,96)||2.00||0.00 |- | 50x SYBG||0.80||80.00 |- | 2x Phusion MM||50.00||5000.00 |- | H2O||37.00||3700.00 |- | Total||100.00||Aliquot 88ul, add 10ul template and 2 ul of AmpR6.3 IndX |} '''Program''' 98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x5 -> (98C 10s -> 72C 20s)x13 -> 72C 3min * Since the first plate didn't give a very good amplification, I check back to my previous note and saw that the PCR profile that I currently used were a little different from the previous protocol. Then I changed the number of cycle in the first loop from 5x to 8x. '''Plate2''' {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|''' Components''' | align="center" style="background:#f0f0f0;"|'''1rxn''' | align="center" style="background:#f0f0f0;"|'''100 rxn mix''' |- | Captured template||10.00||0.00 |- | 10uM AmpF6.4Sol||2.00||200.00 |- | 10uM AmpR6.3 (IndX, X 1,2,3,β¦,96)||2.00||0.00 |- | 50x SYBG||0.80||80.00 |- | 2x Phusion MM||50.00||5000.00 |- | H2O||35.20||3520.00 |- | Total||100.00||Aliquot 88ul, add 10ul template and 2 ul of AmpR6.3 IndX |} '''Program''' 98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x11 -> 72C 3min === Plate layout and barcode primers === {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''A1''' | align="center" style="background:#f0f0f0;"|'''A2''' | align="center" style="background:#f0f0f0;"|'''A3''' | align="center" style="background:#f0f0f0;"|'''A4''' | align="center" style="background:#f0f0f0;"|'''A5''' | align="center" style="background:#f0f0f0;"|'''A6''' | align="center" style="background:#f0f0f0;"|'''A7''' | align="center" style="background:#f0f0f0;"|'''A8''' | align="center" style="background:#f0f0f0;"|'''A9''' | align="center" style="background:#f0f0f0;"|'''A10''' | align="center" style="background:#f0f0f0;"|'''A11''' | align="center" style="background:#f0f0f0;"|'''A12''' |- | AmpR6.3.Ind1||AmpR6.3.Ind9||AmpR6.3.Ind17||AmpR6.3.Ind25||AmpR6.3.Ind33||AmpR6.3.Ind41||AmpR6.3.Ind49||AmpR6.3.Ind57||AmpR6.3.Ind65||AmpR6.3.Ind73||AmpR6.3.Ind81||AmpR6.3.Ind89 |- | B1||B2||B3||B4||B5||B6||B7||B8||B9||B10||B11||B12 |- | AmpR6.3.Ind2||AmpR6.3.Ind10||AmpR6.3.Ind18||AmpR6.3.Ind26||AmpR6.3.Ind34||AmpR6.3.Ind42||AmpR6.3.Ind50||AmpR6.3.Ind58||AmpR6.3.Ind66||AmpR6.3.Ind74||AmpR6.3.Ind82||AmpR6.3.Ind90 |- | C1||C2||C3||C4||C5||C6||C7||C8||C9||C10||C11||C12 |- | AmpR6.3.Ind3||AmpR6.3.Ind11||AmpR6.3.Ind19||AmpR6.3.Ind27||AmpR6.3.Ind35||AmpR6.3.Ind43||AmpR6.3.Ind51||AmpR6.3.Ind59||AmpR6.3.Ind67||AmpR6.3.Ind75||AmpR6.3.Ind83||AmpR6.3.Ind91 |- | D1||D2||D3||D4||D5||D6||D7||D8||D9||D10||D11||D12 |- | AmpR6.3.Ind4||AmpR6.3.Ind12||AmpR6.3.Ind20||AmpR6.3.Ind28||AmpR6.3.Ind36||AmpR6.3.Ind44||AmpR6.3.Ind52||AmpR6.3.Ind60||AmpR6.3.Ind68||AmpR6.3.Ind76||AmpR6.3.Ind84||AmpR6.3.Ind92 |- | E1||E2||E3||E4||E5||E6||E7||E8||E9||E10||E11||E12 |- | AmpR6.3.Ind5||AmpR6.3.Ind13||AmpR6.3.Ind21||AmpR6.3.Ind29||AmpR6.3.Ind37||AmpR6.3.Ind45||AmpR6.3.Ind53||AmpR6.3.Ind61||AmpR6.3.Ind69||AmpR6.3.Ind77||AmpR6.3.Ind85||AmpR6.3.Ind93 |- | F1||F2||F3||F4||F5||F6||F7||F8||F9||F10||F11||F12 |- | AmpR6.3.Ind6||AmpR6.3.Ind14||AmpR6.3.Ind22||AmpR6.3.Ind30||AmpR6.3.Ind38||AmpR6.3.Ind46||AmpR6.3.Ind54||AmpR6.3.Ind62||AmpR6.3.Ind70||AmpR6.3.Ind78||AmpR6.3.Ind86||AmpR6.3.Ind94 |- | G1||G2||G3||G4||G5||G6||G7||G8||G9||G10||G11||G12 |- | AmpR6.3.Ind7||AmpR6.3.Ind15||AmpR6.3.Ind23||AmpR6.3.Ind31||AmpR6.3.Ind39||AmpR6.3.Ind47||AmpR6.3.Ind55||AmpR6.3.Ind63||AmpR6.3.Ind71||AmpR6.3.Ind79||AmpR6.3.Ind87||AmpR6.3.Ind95 |- | H1||H2||H3||H4||H5||H6||H7||H8||H9||H10||H11||H12 |- | AmpR6.3.Ind8||AmpR6.3.Ind16||AmpR6.3.Ind24||AmpR6.3.Ind32||AmpR6.3.Ind40||AmpR6.3.Ind48||AmpR6.3.Ind56||AmpR6.3.Ind64||AmpR6.3.Ind72||AmpR6.3.Ind80||AmpR6.3.Ind88||AmpR6.3.Ind96 |} = PAGE Quantification of Sequencing Libraries = * To purify the seq lib, 100ul of amplified amplicons plate2 were combined with 50ul of plate1 (total volume of amplified amplicon = 150ul) into 96-well plate * Purify seq lib with 0.7 volume Ampure beads, elute with 60ul EB * Quantify DNA concentration of amplified amplicon in 6% TBE PAGE (12-well gel) (load 1ul of amplified amplicon)<br> [[File:ZhangLab_2 2011-08-17 13hr 27min_A1-A10.jpg| 300px]][[File:ZhangLab_2 2011-08-17 13hr 19min_B1-B10PQ.jpg| 300px]] * Pool ~25 ng of each amplified sampled into the same tube (=96X25 ng = 2.4ug) {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Well ID''' | align="center" style="background:#f0f0f0;"|'''conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Total amount in 60ul (ng)''' | align="center" style="background:#f0f0f0;"|'''Volume for 25ng (ul)''' | align="center" style="background:#f0f0f0;"|'''Well ID''' | align="center" style="background:#f0f0f0;"|'''conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Total amount in 60ul (ng)''' | align="center" style="background:#f0f0f0;"|'''Volume for 25ng (ul)''' | align="center" style="background:#f0f0f0;"|'''Well ID''' | align="center" style="background:#f0f0f0;"|'''conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Total amount in 60ul (ng)''' | align="center" style="background:#f0f0f0;"|'''Volume for 25ng (ul)''' |- | A1||7.42||445.24||3.37||C1||5.62||337.48||4.44||E1||7.35||440.82||3.40 |- | A2||5.64||338.19||4.44||C2||4.60||276.17||5.43||E2||6.74||404.16||3.71 |- | A3||4.53||271.61||5.52||C3||3.07||184.27||8.14||E3**||0.19||11.46||60** |- | A4||4.10||245.97||6.10||C4||2.64||158.13||9.49||E4||3.41||204.74||7.33 |- | A5||4.34||260.26||5.76||C5||0.95||56.71||26.45||E5||1.90||114.02||13.16 |- | A6||4.37||262.10||5.72||C6||4.85||290.89||5.16||E6||1.36||81.30||18.45 |- | A7||3.64||218.44||6.87||C7||4.26||255.41||5.87||E7||3.26||195.32||7.68 |- | A8||9.62||577.08||2.60||C8||2.28||136.64||10.98||E8||2.49||149.17||10.06 |- | A9||5.87||352.21||4.26||C9||0.65||39.17||38.30||E9||1.36||81.74||18.35 |- | A10||7.38||442.57||3.39||C10||2.01||120.65||12.43||E10||2.20||132.04||11.36 |- | B1||5.84||350.35||4.28||D1||5.56||333.37||4.50||F1||6.67||400.42||3.75 |- | B2||5.46||327.84||4.58||D2||4.53||271.74||5.52||F2||3.02||181.16||8.28 |- | B3||2.03||121.68||12.33||D3||1.88||112.91||13.28||F3||3.12||187.27||8.01 |- | B4||2.88||172.65||8.69||D4||2.64||158.53||9.46||F4||2.70||162.27||9.24 |- | B5||3.77||226.31||6.63||D5||1.90||114.20||13.14||F5**||0.10||6.08||60** |- | B6||3.43||205.99||7.28||D6||2.82||169.46||8.85||F6||2.18||130.98||11.45 |- | B7||4.04||242.53||6.18||D7||2.95||177.20||8.47||F7||1.20||71.81||20.89 |- | B8||3.92||235.42||6.37||D8**||-0.45||-26.73||60**||F8||0.73||43.78||34.26 |- | B9||3.46||207.79||7.22||D9||1.52||91.15||16.46||F9||0.72||43.22||34.71 |- | B10||0.58||34.94||42.93||D10||3.01||180.35||8.32||F10||4.79||287.50||5.22 |}<br> {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Well ID''' | align="center" style="background:#f0f0f0;"|'''conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Total amount in 60ul (ng)''' | align="center" style="background:#f0f0f0;"|'''Volume for 25ng (ul)''' | align="center" style="background:#f0f0f0;"|'''Well ID''' | align="center" style="background:#f0f0f0;"|'''conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Total amount in 60ul (ng)''' | align="center" style="background:#f0f0f0;"|'''Volume for 25ng (ul)''' |- | G1||5.54||332.34||4.51||A11||2.96||177.41||8.45 |- | G2||4.10||245.73||6.10||B11||3.01||180.38||8.32 |- | G3||3.70||222.02||6.76||C11||3.71||222.85||6.73 |- | G4||0.89||53.24||28.17||D11||0.54||32.69||45.88 |- | G5||1.61||96.85||15.49||E11||1.33||80.08||18.73 |- | G6||3.74||224.69||6.68||F11||3.86||231.73||6.47 |- | G7||2.78||166.71||9.00||G11||3.33||199.56||7.52 |- | G8||1.18||70.72||21.21||H11||4.59||275.44||5.45 |- | G9||0.80||48.13||31.16||A12||6.58||394.93||3.80 |- | G10||3.28||196.77||7.62||B12||4.96||297.75||5.04 |- | H1||5.58||334.76||4.48||C12||5.13||307.60||4.88 |- | H2||3.56||213.89||7.01||D12||4.86||291.87||5.14 |- | H3||4.43||266.06||5.64||E12||5.25||314.85||4.76 |- | H4||1.13||67.98||22.07||F12||4.58||274.81||5.46 |- | H5||2.94||176.59||8.49||G12||6.25||375.00||4.00 |- | H6||3.21||192.76||7.78||H12||3.27||196.30||7.64 |- | H7||1.73||103.53||14.49|||||||| |- | H8||6.92||415.47||3.61|||||||| |- | H9||2.34||140.57||10.67|||||||| |- | H10||6.04||362.62||4.14|||||||| |}<br> * Perform PAGE size selection in 6% TBE PAGE (use ~ 4 of 1mm 5-well gel to prevent overloading of DNA )<br> [[File:ZhangLab_2 2011-08-18 12hr 47min_GA-SeqLibPAGE SS-1.jpg| 450px]]<br> * Purify DNA from acrylamide gel as previously described * After precipitation and resuspen sequencing DNA with H2O ~ 100 ul, quantify DNA conc. by either PAGE quantification and Qubit dsDNA assay.<br> * 0.6 and 0.3ul of sequencing libraries were analyzed by PAGE quantification, conc.: 9.65 ng/ul or 42.3nM (yields = 9.65 ng/ul x 100ul = 965ng) [[File:ZhangLab_2 2011-08-19 11hr 21min_PQ-SeqLib.jpg| 250px]]<br> * Concentration by Qubit ds-DNA HS, conc.: 6.61ng/ul or 28.9 nM (yields = 6.61ng/ul x 100ul = 661ng) * Used the concentration from Qubit as the reference and the sequencing libraries are ready to be sequenced by HiSeq. * Sequencing: Sample ID: GA-plate1.Ind1-96.SeqLib_Aug9,2011: at BIOGEM core-->2011_09_22 * Diluted sequencing library to 10nM volume 120ul --> Mix 41.52ul of 28.9nM seqlib with 78.48ul H2O
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