ContentsIntroduction...........................................2Overview.............................................2StorageandStability.....................................2KitContents...........................................3MaterialstoBeProvidedbyUser.............................3BeforeStarting.........................................4E.Z.N.A.WaterDNAKitProtocol............................5®TroubleshootingGuide....................................7IntroductionTheE.Z.N.A.WaterDNAKitallowsrapidandreliableisolationofhigh-quality®DNAfromvariousmicroorganismsinwatersamples.Thesystemcombinesthereversiblenucleicacid-bindingpropertiesofHiBindmatrixwiththespeed®andversatilityofspincolumntechnologytoeliminatePCRinhibitingcompoundsfromwatersample.PurifiedDNAissuitableforPCR,restrictiondigestion,andhybridizationtechniques.Therearenoorganicextractionsthusreducingplasticwasteandhands-ontimetoallowmultiplesamplestobeprocessedinparallel.OverviewIfusingtheE.Z.N.A.WaterDNAKitforthefirsttime,pleasereadthis®booklettobecomefamiliarwiththeprocedure.Watersampleisfirstfilteredusingmicroporousfilter.Thefilteristhenputintoatubecontainsbeadsandbuffertohomogenizeandlysisthesample.Humicacid,proteins,polysaccharides,andothercontaminantsaresubsequentlyprecipitatedafteraheat-frozenstep.ContaminantsarefurtherremovedbyaspecialHTRreagenttreatment.BindingconditionsarethenadjustedandthesampleisappliedtoanHiBindDNAspin-column.Tworapidwashstepsremovetrace®contaminantsandpureDNAiselutedinwaterorlowionicstrengthbuffer.PurifiedDNAcanbedirectlyusedindownstreamapplicationswithouttheneedforfurtherpurification.StorageandStabilityAllcomponentsoftheE.Z.N.A.WaterDNAKitshouldbestoredat22C-25C.®ooUnderthisstorageconditions,DNAhassuccessfullybeenpurifiedandusedforPCRafter24monthsofstorage.Duringshipment,orstorageincoolambientconditions,precipitatesmayforminsomebuffers.Itispossibletodissolvesuchdepositsbyincubationthesolutionat65C.o2KitContentsProductNumberD5525-00D5525-01D5525-02Purificationtimes5Preps50Preps200PrepsHiBindDNAColumns550200®2mLCollectionTubes10100400GlassBeads2.7g27g120gHTRReagent1mL10mL40mLBufferSLX18mL180mL3x220mLSP2Buffer6mL60mL220mLXP1Buffer5mL40mL180mLElutionBuffer5mL30mL100mLDNAWashBuffer2mL20mL2×50mLInstructionBooklet111Materialstobeprovidedbyuser!Microcentrifugecapableofatleast14,000xg!tabletopcentrifugecapableof4000xg.!adapterfor50mlcentrifugetube.!Nuclease-free1.5mLor2mLmicrofugetubes!Nuclease-free50mlcentrifugetubecapableof4000xg.!Waterbathequilibratedto65Co!Absolute(96%-100%)ethanol!RNaseAstocksolutionat25mg/mL3BeforeStarting!PleasereadtheentirebooklettobecomefamiliarwiththeE.Z.N.A.®WaterDNAKitprotocol.!DiluteDNAWashBufferConcentratewithabsoluteethanolasfollowsandstoreatroomtemperature.D5525-00Add8mL(96%-100%)ethanol.D5525-01Add80mL(96%-100%)ethanoltoeachbottle.D5525-02Add200mL(96%-100%)ethanoltoeachbottle.!PreheatElutionBufferat65CoWaterDNAIsolationProtocol1.Filterthewatersamplesusingamicroporousfilterpaper(0.22ìmor0.45ìm).Thevolumeofwatercanbeuseddependonthemicrobialloadandturbidityofthewatersample.Forturbidwatersample,itishighlyrecommendedtouseaprefilterpapertopreventcloggingofthemicroporousfilter.2.Takethefiltermembranefromthefilteradapterandscissorthemembraneforquartering.Insertthefiltermembranetoanascetical50mlcentrifugetube.3.Add3mlSLXBufferand500mgglassbeads(suppplied)to...
