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1、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;italsohelpsr
2、emoveproteinsandlipidsinthecell.Ethylenediaminetetraaceticacid(EDTA),achelatortoremovemetalionsinsolutiontoprevenDNasefromcuttinguptheDNA.RNaseisalsopresentinthebufferatthisstep,tobreakuptheRNApresentinthecells.2)RemoveproteinProteinaseK,itremainsactiveatelevatedt
3、emperatures,sothesolutioncanbeheatedtoabout55°Ctoaidproteininactivationandremovalbythedetergent.3)ExtractDNAfrombufferOncethecellsarebrokenopenandtheRNA,proteins,andlipidshavebeendissolvedinthebuffer,theDNAmustbeseparatedfromthesematerials.Phenol:removetheproteins
4、,leavingDNAandotherwater-solublematerialsbehindbycentrifugation.TheDNAisthenextractedfromthewaterphaseusingchloroformandprecipitatedfromthechloroformusingethylalcoholmixedwithsodiumacetatesalt.4)DNAprecipitationTheethanolcanprecipitateDNAfromwaterphase2.Materialsa
5、ndSolutionsAllreagentsareprecooledorkeptat4°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/LNaCl1)Extractionsolution(ES)(1
6、00mMEDTA,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)1.666ml83.3μlDDWater498.3ml25ml1.Experimentalprotocol1)HarvestcellsandwashcellswithPBS
7、(~106cells)2)Suspendcellsinto500μlES3)Slowlyadd10μlproteasesK(5mg/ml,Finalconcentrationof100μg/ml)totheabovecellsuspensionwhilegentlymixing.Incubatethissolutionat55°Cforaminimumof2-3hwithoccasionalmanualormechanicalgentlemixing.4)Anequalvolumeofphenol(500μl)isadde
8、dtothecelllysate.Centrifugeat12,000rpmfor5mintoseparatethetwophases.Theaqueous(top)phaseistransferredtoanewtubeusingawideboretransferpipette.Note:cutati
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