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==DMR220K Normalization Capture== * gDNA template: bis-cvt BJ-iPS (145.5ng/ul, Nanodrop) ===Calculations=== {| {{table}} border=1 | align="center" style="background:#f0f0f0;"|Probe/Target Ratio||100|| 100 || 100 |- | align="center" style="background:#f0f0f0;"|Probes size||6500|| 27000 || 55000 |- | align="center" style="background:#f0f0f0;"|Template (ng)||200|| 200 || 200 |- | align="center" style="background:#f0f0f0;"|Human gDNA MW (g/mole)||1.82E+012|| 1.82E+012|| 1.82E+012 |- | align="center" style="background:#f0f0f0;"|Human gDNA(moles)||1.10E-019|| 1.10E-019|| 1.10E-019 |- | align="center" style="background:#f0f0f0;"|Probes required (moles)||1.10E-017|| 1.10E-017|| 1.10E-017 |- | align="center" style="background:#f0f0f0;"|Probes MW (g/mole)||2.03E+008|| 8.45E+008 || 1.72E+009 |- | align="center" style="background:#f0f0f0;"|Amount probes (ng)||2.23|| 9.27 || 18.88 |} ===Probes Subset Information=== * Probes were prepared by DD and ZL. * We re-quantified the probes and got higher concentration, the issue was probably not shaking in SYBR Gold solution long enough. {| {{table}} border=1 | align="center" style="background:#f0f0f0;"|'''Subset ID''' | align="center" style="background:#f0f0f0;"|'''Size''' | align="center" style="background:#f0f0f0;"|'''Conc(ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Vol(ul)''' | align="center" style="background:#f0f0f0;"|'''Est. Efficiency Coefficient''' |- | DMR.s1||6500||20.14||12||1106.55 |- | DMR.s2||6500||25.32||12||597.53 |- | DMR.s3||55000||37.42||12||226.35 |- | DMR.s4||55000||52.65||13||56.42 |- | DMR.s5||55000||61.93||13||11.68 |- | DMR.s6||27000||69.03||19||1.26 |- | DMR.s7||27000||44.27||19||1 |} [[File:DMR220K_cap_norm..png | 200px]] [[File:ZhangLab_2_2010-09-23_DMR220_subset_quant.png | 400px]] ===Multiplexing Setup=== * We calculated two multipliers for the multiplexing setup * A. Using subset 4 as baseline 1 and the ratios of the estimated efficiencies of other probe sets as multiplier. * B. For 1-3, use 10x of the multiplier in A, and for 5-7, use 0.5x of the multiplier in A. {| {{table}} border=1 | align="center" style="background:#f0f0f0;"|'''Probes subset ID''' | align="center" style="background:#f0f0f0;"|'''Multiplier A''' | align="center" style="background:#f0f0f0;"|'''Amount ProbesxMultiplier''' | align="center" style="background:#f0f0f0;"|'''Vol Req.(ul)''' | align="center" style="background:#f0f0f0;"|'''Multiplier B''' | align="center" style="background:#f0f0f0;"|'''Amount ProbesxMultiplier''' | align="center" style="background:#f0f0f0;"|'''Vol Req.(ul)''' |- | DMR.s1||0.05||0.11||0.01||0.51||1.14||0.06 |- | DMR.s2||0.09||0.21||0.01||0.94||2.11||0.08 |- | DMR.s3||0.25||4.71||0.13||2.49||47.07||1.26 |- | DMR.s4||1||18.88||0.36||1||18.88||0.36 |- | DMR.s5||4.83||91.22||1.47||2.42||45.61||0.74 |- | DMR.s6||44.78||415.09||6.01||22.39||207.55||3.01 |- | DMR.s7||56.42||523.01||11.81||28.21||261.51||5.91 |} ===Experimental Setup=== * We prepared the reactions in PCR tubes * Buffer used is 10x Ampligase Buffer * First 7 reactions: Capture with individual probe set. {| {{table}} border=1 | align="center" style="background:#f0f0f0;"|'''Dilution factor''' | align="center" style="background:#f0f0f0;"|'''Individual Capture Rxn''' | align="center" style="background:#f0f0f0;"|'''Probes''' | align="center" style="background:#f0f0f0;"|'''Template''' | align="center" style="background:#f0f0f0;"|'''Buffer''' | align="center" style="background:#f0f0f0;"|'''H2O''' | align="center" style="background:#f0f0f0;"|'''Total''' |- | 0.01||DMR.s1||11.08||1.37||2||5.54||20 |- | 0.01||DMR.s2||8.81||1.37||2||7.81||20 |- | 1||DMR.s3||0.5||1.37||2||16.12||20 |- | 0.1||DMR.s4||3.59||1.37||2||13.04||20 |- | 0.1||DMR.s5||3.05||1.37||2||13.58||20 |- | 0.1||DMR.s6||1.34||1.37||2||15.28||20 |- | 0.1||DMR.s7||2.09||1.37||2||14.53||20 |} * Multiplex A: Capture with mixed probes. {| {{table}} border=1 | align="center" style="background:#f0f0f0;"|'''Dilution factor''' | align="center" style="background:#f0f0f0;"|'''Multiplex A''' | align="center" style="background:#f0f0f0;"|'''Probes vol''' | align="center" style="background:#f0f0f0;"|'''Template''' | align="center" style="background:#f0f0f0;"|'''Buffer''' | align="center" style="background:#f0f0f0;"|'''H2O''' | align="center" style="background:#f0f0f0;"|'''Total''' |- | 0.01||DMR.s1||0.56|||||||| |- | 0.01||DMR.s2||0.83|||||||| |- | 0.1||DMR.s3||1.26|||||||| |- | 0.1||DMR.s4||3.59|||||||| |- | 1||DMR.s5||1.47|||||||| |- | 1||DMR.s6||6.01|||||||| |- | 1||DMR.s7||11.81|||||||| |- | ||||align="center" style="background:#f0f0f0;"|25.54||align="center" style="background:#f0f0f0;"|1.37||align="center" style="background:#f0f0f0;"|3||align="center" style="background:#f0f0f0;"|0.09||align="center" style="background:#f0f0f0;"|30 |} * Multiplex B: Capture with mixed probes. {| {{table}} border=1 | align="center" style="background:#f0f0f0;"|'''Dilution factor''' | align="center" style="background:#f0f0f0;"|'''Multiplex B''' | align="center" style="background:#f0f0f0;"|'''Probes vol''' | align="center" style="background:#f0f0f0;"|'''Template''' | align="center" style="background:#f0f0f0;"|'''Buffer''' | align="center" style="background:#f0f0f0;"|'''H2O''' | align="center" style="background:#f0f0f0;"|'''Total''' |- | 0.01||DMR.s1||5.65|||||||| |- | 0.01||DMR.s2||8.32|||||||| |- | 1||DMR.s3||1.26|||||||| |- | 1||DMR.s4||0.36|||||||| |- | 1||DMR.s5||0.74|||||||| |- | 1||DMR.s6||3.01|||||||| |- | 1||DMR.s7||5.91|||||||| |- | ||||align="center" style="background:#f0f0f0;"|25.24||align="center" style="background:#f0f0f0;"|1.37||align="center" style="background:#f0f0f0;"|3||align="center" style="background:#f0f0f0;"|0.39||align="center" style="background:#f0f0f0;"|30 |} ===RT PCR=== {| {{table}} border=1 | align="center" style="background:#f0f0f0;"|'''Reagent''' | align="center" style="background:#f0f0f0;"|'''Concentration''' | align="center" style="background:#f0f0f0;"|'''Vol (ul)''' | align="center" style="background:#f0f0f0;"|'''Final Concentration''' | align="center" style="background:#f0f0f0;"|'''11x Master Mix''' |- | Template||---||10|||| |- | AmpR6.3IndX||10uM||2||0.2uM||22 |- | AmpF6.3Sol||10uM||2||0.2uM|| |- | Phusion HF 2x MM||2x||50||1x||550 |- | SYBR Green||50x||0.4||1x||4.4 |- | H2O||---||36||||396 |- | ||TOTAL||100.4||||972.4 |} '''Program''' 98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min Purified each reaction with 0.7x AMPure Beads. [[File:ZhangLab 2 2010-10-07 DMR220KNorm.jpg | 300px]] 1 ul of each subset loaded. We combined s1-s7, A, and B to form the barcoded DMR library, then performed PAGE purification. ===Q-PCR === {| {{table}} border=1 | align="center" style="background:#f0f0f0;"|'''Reagent''' | align="center" style="background:#f0f0f0;"|'''Concentration''' | align="center" style="background:#f0f0f0;"|'''Vol (ul)''' | align="center" style="background:#f0f0f0;"|'''Final Concentration''' | align="center" style="background:#f0f0f0;"|'''10x Master Mix''' |- | Template||---||3|||| |- | AmpR6.3||100uM||0.6||0.2uM||6 |- | AmpF6.3||100uM||0.6||0.2uM||6 |- | Phusion HF 2x MM||2x||750||1x||750 |- | SYBR Green||50x||1.2||1x||12 |- | H2O||---||72||||720 |- | ||TOTAL||152.4||||1524 |} '''Results''' {| {{table}} border=1 | Subset||Mean CT||Ratio compared to s1||Probes size||Adjusted Ratio||% of s1 |- | s1||19.51||1||6500||1||100% |- | s2||20.33||0.57||6500||0.57||57% |- | s3||16.99||5.76||55000||0.68||68% |- | s4||18.8||1.64||55000||0.19||19% |- | s5||20.45||0.52||55000||0.06||6% |- | s6||18.91||1.52||27000||0.36||36% |- | s7||22||0.18||27000||0.04||4% |} '''PAGE''' We repeated the Q PCR but stopping at cycle no. 22. Then ran a page gel to see which products are being amplified. [[File:ZhangLab 2 2010-10-08 17hr 36min DMR220k S1-7 MixAB 21cycles.jpg|500px]]
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