資源描述：
《《核酸研究方法》ppt課件》由會(huì)員上傳分享，免費(fèi)在線閱讀，更多相關(guān)內(nèi)容在教育資源-天天文庫(kù)。
1、第十五章核酸的研究方法1、核酸的分離、提純和定量測(cè)定DNA的分離RNA的分離核酸的定量IsolationofNucleicAcidsGoals:removalofproteinsDNAvsRNAisolationofaspecifictypeofnucleicacidTypesofMethods:differentialsolubility‘a(chǎn)dsorption’methodsdensitygradientcentrifugationTypesofDNA:genomic(chromosomal)organellar(satellite)plasmid(extra-chromosomal)p
2、hage/viral(dsorss)complementary(mRNA)GeneralFeatures:denaturingcelllysis(SDS,alkali,boiling,chaotropic)?enzymetreatmentsproteaseRNase(DNase-free)DNase(RNase-free)CollectionofcellsUsesoftbrushestodislodgeepithelialcellsliningthemouth.Amplecellcollectionisverycriticalforsuccess.What’sinLysisBuffer?T
3、risbuffertomaintainpHofsolutionsothatDNAisstableSDStodissolvemembranesofcell,allowingDNAtobereleasedintosolutionSDSalsodenaturesproteins,makingthemmoresusceptibletoproteasecleavageLysisbufferOSOOO-SDSCH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3-WhyaddProtease?Proteasedestroysnuclearproteinsthat
4、bindDNA,andcytoplasmicenzymesthatbreakdownanddestroyDNA(nucleases)ProteasetreatmentincreasestheyieldofDNAAddingsalt(5MNaCl)Na+bindstophosphategroupsofDNA,neutralizingtheelectricchargeofDNANaClallowsDNAmoleculesaggregateinsteadofrepellingeachother,makingiteasierforDNAtoprecipitateoutofsolutionwhena
5、lcoholisaddedAddingicecoldalcoholDNAcannotdissolveinalcoholTheadditionofcoldalcoholmakestheDNAclumptogetherandprecipitateoutofsolutionPrecipitatedDNAmoleculesappearaslongpiecesoffluffy,stringy,web-likestrandsMicroscopicoxygenbubbles“aggregate”or“fuse”together,simultaneouslywiththeDNAprecipitationT
6、helarger,visibleairbubblesactto“l(fā)ift”theDNAoutofsolution,fromtheaqueousintotheorganicphaseDNAPrecipitationHighMWGenomicDNAIsolationTypicalProcedureCellLysis0.5%SDS+proteinaseK(55oseveralhours)PhenolExtractiongentlerockingseveralhoursEthanolPrecipitationRNAsefollowedbyproteinaseKRepeatphenolextrac-
7、tionandEtOHpptPhenolExtractionmixsamplewithequalvolumeofsat.phenolsolnretainaqueousphaseoptionalchloroform/isoamylalcoholextraction(s)?aqueousphase(nucleicacids)?phenolphase(proteins)HighMWGenomicDNAIsolationTypi
