E.coli. plasmid purification-Minipreps

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1、PurificationofplasmidDNAPlasmidsaresmalldouble-strandedDNApiecesofDNAthatexistoutsidethechromosomeofthecellinwhichtheyarelocated.Almostallplasmidsarecircular.Theycontainasmallnumberofgenes,whichcanincludegenesthatmakecellsresistanttoantibiotics.Plasmidsareusedextensive

2、lyincloningDNA.YouinsertsomeforeignDNAintotheplasmidDNA,andtheninfectcellswiththeDNAinaprocesscalledtransformation.Thereareseveralwaystopurifyplasmids.Thesemethodsareoftencalled“minipreps”becauseyouarepurifyingDNAfromasmallvolumeofcells.Miniprepsinvolvelysingthecellsan

3、dpurifyingtheDNAviacentrifugationand/ormembranebinding.Themethodwewilldousesasilica-gelmembranetobindtheDNA,whichhasbeendevelopedbythecompanyQiagen.YouwillpurifytheplasmidDNAandanalyzeit.Welysethecellsusingamodifiedalkalinelysisprocedure.ThistechniquewasinventedbyBirnb

4、oimandDoly.Othertypesofminiprepsmayuseadifferentprocedureforlysingthecells.Ingeneral,inthealkalinelysismethod,thecellsarebroughttoahighpHtonotonlylysethecells,butalsotodenaturetheDNA.TheDNAsolutionisthenneutralized.SinceplasmidDNAiscircularandsupercoiled,whenthepHisbro

5、ughtbackdowntoneutral,theplasmidDNAsnapsbacktobeingdouble-stranded.Bycontrast,genomicDNAissolargethatitisbrokenintolinearpiecesnomatterwhatwedo.ThelinearDNAdenaturesinalkaliandformsprecipitateswhenthepHislowered.Theprecipitateisremovedviacentrifugation.Thesupernatantis

6、thenappliedtothesilica-gelmembranetofurtherpurifytheplasmidDNA.Undertherightconditionsdouble-strandplasmidDNAstickstothemembranewhilesingle-strandDNAandRNAdonot.ThemembranedoesnotrequiretheuseoforganicsolventsandletsuseasilywashtheDNAtoremovecontaminants.Wethenelutethe

7、DNAfromthemembranebyusingalow-ionicstrengthbuffer.TheplasmidDNAthatweobtainisverypure,andcanberestrictedorusedinDNAsequencing.MaterialsDNApurification1.5mlculturesofplasmidinfectedE.coli,grownovernightinTB(Terrificbroth)TEbuffer(10mMTris-HCl,pH8.0,1mMEDTA)BufferP1Buffe

8、rP2BufferN3BufferPBBufferPEBufferEB:10mMTris-HCl,pH8.5QIAprepspincolumns.Microfugetubes(1.7ml)Collectiontubes:2mlcapl