EpiTectBisulfite ConversionProcedure亚硫酸盐转化DNAand EpiTectBisulfite reactionreaction buffersDNAconversioninthermal cycler (5h)Transferto1.5mlmicrofubeAddBufferBLWCm-DLoadSpinColumnBindffffWashAddBufferBW纯化DesulfonationAdd Buffer BDWash (x 2)AddBufferBWElute
亚硫酸盐转化纯化
DNA亚硫酸盐转化1.Add8o0μl RNase-freewaterto eachaliguotof BisulfiteMixneeded,andvortex until Bisulfite Mix is completely dissolved. This may take up to 5 min.Dissolving the Bisulfite Mix may require heating the solution to 60°C.2.Set up the bisulfite reactions in 200 μl PCR tubes according to Table 1.AddeachcomponentintheorderlistedTable1.BisulfitereactionsetupHigh concentration samplesLowconcentrationsamples(1 ng - 500 ng)(1ng - 2 μg)ComponentVolumeper reaction (μl)Volumeper reaction (μl)DNA solutionVariablet (maximum40 μl)Variable*(maximum20μl)VariabletVariable*RNase-free water8585Bisulfite Mix3515DNAProtectBuffer140140Totalvolume*ThecombinedvolumeofDNA solutionandRNase-freewatermusttotal2Oμl.tThe combined volumeof DNA solution and RNase-free water must total 40 μl
DNA亚硫酸盐转化
DNA亚硫酸盐转化3.ClosethePCRtubes andmixthebisulfitereactionsthoroughly.DNAProtectBuffer should turn blue indicating sufficient mixing and correct pH.4.Programthethermal cycleraccordingtoTable2.Useacyclerwithaheated lid.Ifusingathermal cyclerthatdoesnotallowyoutoenterthereactionvolume(140μl),settheinstrumenttothelargestvolume settingavailable.Table2.BisulfiteconversionthermalcyclerconditionsStepTimeTemperature95℃5 minDenaturation60℃Incubation25 min5 min95℃Denaturation60℃Incubation85min(1h25min)95℃Denaturation5min60℃Incubation175min(2h55min)Hold20℃Indefinite*
DNA亚硫酸盐转化
DNA纯化1.For startingmaterial<10OngDNA,adddissolvedcarrierRNAtoBufferBL.Prepareonlythe amountneeded forthe numberof reactions (Table1)2.Upon completionof thebisulfite conversion (Protocol 1),brieflycentrifugethePCRtubes.Transferthereactionstoclean1.5ml microcentrifugetubes3.Add310ulBufferBL(with10μg/mlcarrierRNAfor<100ngDNA;step1toeach sample.Mixbyvortexing andthen centrifugebriefly4.Add 250 μl ethanol (96-100%) to each sample.Mix by pulse vortexing for15sandthencentrifugebrieflytoremovedropsfrominsidethelid.5.PlaceMinElute?DNA spin columns and collectiontubes inarack.Transferthe entire contents of eachtube (step 4)to a corresponding spin column.6.Centrifugethespincolumnsatmaximumspeedfor1min.Discardtheflow-throughandplacethespin columns backintothecollectiontubes
DNA纯化