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Chapter1DNAextraction说明：本原理及方法是个人整理，用来给研究生教学用的，用本方法可以提取到理想的DNA。各实验室提供的细胞裂解液浓度各有不同，只要经过实验证实的，都可以用来提取到理想的DNA.1.ExperimentalPrinciples1)Celllysis(lysisbuffer,containingSDS,EDTA,Tris-HCl,andRNase)SDS,adetergentisaddedtothebuffertobreakopenthecellmembranes;italsohelpsremoveproteinsandlipidsinthecell.Ethylenediaminetetraaceticacid(EDTA),achelatortoremovemetalionsinsolutiontoprevenDNasefromcuttinguptheDNA.RNaseisalsopresentinthebufferatthisstep,tobreakuptheRNApresentinthecells.2)RemoveproteinProteinaseK,itremainsactiveatelevatedtemperatures,sothesolutioncanbeheatedtoabout55°Ctoaidproteininactivationandremovalbythedetergent.3)ExtractDNAfrombufferOncethecellsarebrokenopenandtheRNA,proteins,andlipidshavebeendissolvedinthebuffer,theDNAmustbeseparatedfromthesematerials.Phenol:removetheproteins,leavingDNAandotherwater-solublematerialsbehindbycentrifugation.TheDNAisthenextractedfromthewaterphaseusingchloroformandprecipitatedfromthechloroformusingethylalcoholmixedwithsodiumacetatesalt.4)DNAprecipitationTheethanolcanprecipitateDNAfromwaterphase2.MaterialsandSolutionsAllreagentsareprecooledorkeptat4°Cbeforeuse.1)ProteinaseK2)PhenolsaturatedwithTE(pH8.0)3)Chloroform4)Isoamylalcohol5)RNasestock(30mg/ml,CatalogNo.R4642-10MG,Sigma)6)10%SDS7)0.5mol/LEDTA,PH=8.08)1mol/LTris-HCl,PH=8.09)1mol/LNaCl10)Extractionsolution(ES)(100mMEDTA,200mMNaCI,50mMTris-HCI(pH8.0),0.5%SDS,50μg/mlRNase)1L50ml0.5mol/LEDTA,PH=8.0200ml10ml1mol/LTris-HCl,PH=8.050ml2.5ml1mol/LNaCl200ml10ml10%SDS50ml2.5mlRNasestock(30mg/ml)1.666ml83.3μlDDWater498.3ml25ml3.Experimentalprotocol1)HarvestcellsandwashcellswithPBS(~106cells)2)Suspendcellsinto500μlES3)Slowlyadd10μlproteasesK(5mg/ml,Finalconcentrationof100μg/ml)totheabovecellsuspensionwhilegentlymixing.Incubatethissolutionat55°Cforaminimumof2-3hwithoccasionalmanualormechanicalgentlemixing.4)Anequalvolumeofphenol(500μl)isaddedtothecelllysate.Centrifugeat12,000rpmfor5mintoseparatethetwophases.Theaqueous(top)phaseistransferredtoanewtubeusingawideboretransferpipette.Note:cutatipusingscissorsorbladetogetawideborepipette.5)AddanequalvolumeofPhenol/chloroform/isoamylalcohol(500μl)intotheaqueousphase,andcentrifugeat12,000rpmfor5mintoseparatethetwophases.Theaqueous(top)phaseistransferredtoanewtube.6)Addanequalvolumeofchloroform(~500μl)intotheaqueousphase,andcentrifugeat12,000rpmfor5mintoseparatethetwophases.Theaqueous(top)phaseistransferredtoanewtube.7)Add2volumeofabsoluteethanol(~900μl)totheaqueousphaseandmixgently.Keepat-20°Cfor30min,andcentrifugeat12,000gfor5min.DNApelletshouldbewashedwith70%ethanoltodecreaseresidualsaltandbrieflydriedundervacuumorair-driedat37°Ctoevaporatetheethanol.8)Theethanol-freeDNAisdissolvedin50μlTE(pH=8.0)orDDwater.Note:TogethigherconcentrationofDNAsolution,addsmallervolumeTEorwater.9)MeasuretheabsorbanceofDNAsolutionat260and280nm.ThepuritycanbeestimatedfromtheratioofA260/A280.Aratioof1.8-2.0suggestsminimalproteincontamination.10)TheDNAsolutionisbeststoredat4°Cor-20°C.QuickGuideforTraditionalDNAExtractiontechnologyQuickGuideforDNAExtractionKitAppendix1:Protocolforremovalofparaffin1)Placeasmallsection(notmorethan25mg)ofparaffin-embeddedtissueina2mlmicrocentrifugetube(notprovided).2)Add1200μlxylene.Vortexvigorously.3)Centrifugeatfullspeedfor5minatroomtemperature.4)Removesupernatantbypipetting.Donotremoveanyofthepellet.5)Add1200μlethanol(96–100%)tothepellettoremoveresidualxyleneandmixgentlybyvortexing.6)Centrifugeatfullspeedfor5minatroomtemperature(15–25°C).7)Carefullyremovetheethanolbypipetting.Donotremoveanyofthepellet.8)Repeatstepsstep5–7once.9)Incubatetheopenmicrocentrifugetubeat37°Cfor10–15minuntiltheethanolhasevaporated.10)Resuspendthetissuepelletinlysisbuffer.Appendix2:Protocolfortissueonglassslides1)Addadropofabsoluteethanolonslide2)Scratchthetissueandtransfertoa2mlmicrotube3)Evaporatetheethanolintheairatroomtemperature4)Addlysisbufferintotube5)Continuestep3ofprotocolforDNAextractionfromculturedcellsNote:1)Tissuefrom1slideisenoughtoextractgenomicDNA.2)Incubationtimefortissuelysisshouldtoaslongaspossible，uptoovernight.