QIAgen病毒DN;RNA共提取试剂盒QIAamp-MinElute-Virus-Spin-Handbook

QIAamp®MinElute®VirusSpinHandbookForsimultaneouspurificationofviralRNAandDNAfromplasma,serum,andcell-freebodyfluidsThirdEditionApril2010Sample&AssayTechnologies1063023_HB19.04.201014:49UhrSeite1Trademarks:QIAGEN®,QIAamp®,QIAcube®,BioRobot®,EZ1™MinElute®(QIAGENGroup);Corex®(Corning,Inc.);Eppendorf®(Eppendorf-Netheler-HinzGmbH).©2002–2007QIAGEN,allrightsreserved.QIAGENisamemberoftheForestStewardshipCouncil(FSC).Fortheproductionofprintedmaterials,includinghandbooks,QIAGENhasapolicytoselectsuppliersthatcomplywithFSCstandardsforprintingprocessesandwell-managedforests.1063023_HB19.04.201014:49UhrSeite2QIAampMinEluteVirusSpinHandbook04/20103ContentsKitContents4Storage4QualityControl5ProductUseLimitations5ProductWarrantyandSatisfactionGuarantee6TechnicalAssistance6SafetyInformation7Introduction8Principleandprocedure8Yieldandsizeofviralnucleicacids10CarrierRNA12Additionofinternalcontrols12EquipmentandReagentstoBeSuppliedbyUser13ImportantNotes14HandlingofQIAampMinEluteColumns14Centrifugation14ProcessingQIAampMinEluteColumnsinamicrocentrifuge14PreparationofRNA15Samplestorage15PreparationofQIAGENProtease15AdditionofcarrierRNAtoBufferAL16BufferAW117BufferAW218Elutionofnucleicacids18ProtocolPurificationofViralNucleicAcidsfromPlasmaorSerum19TroubleshootingGuide22Appendix25OrderingInformation271063023_HB19.04.201014:49UhrSeite3QIAampMinEluteVirusSpinHandbook04/20104KitContentsQIAampMinEluteVirusSpinKit(50)Catalogno.57704Numberofpreps50QIAampMinEluteColumns50CollectionTubes(2ml)200BufferAL*12mlBufferAW1*(concentrate)19mlBufferAW2†(concentrate)13mlBufferAVE†(tubeswithpurplecaps)5x2mlProteaseResuspensionBuffer†6mlCarrierRNA(tubeswithredcaps)310µgQIAGEN®Protease‡1vialHandbook1*Containsachaotropicsalt.Takeappropriatelaboratorysafetymeasuresandweargloveswhenhandling.Notcompatiblewithdisinfectantscontainingbleach.Seepage7forsafetyinformation.†Containssodiumazideasapreservative.‡Resuspensionvolume1.4ml.See“PreparationofQIAGENProtease”,page15.StorageQIAampMinElutecolumnsshouldbestoredat2–8°Cuponarrival.Allbufferscanbestoredatroomtemperature(15–25°C).LyophilizedcarrierRNAcanbestoredatroomtemperature(15–25°C)untiltheexpirationdateonthekitbox.CarrierRNAcanonlybedissolvedinBufferAVE;dissolvedcarrierRNAshouldbeimmediatelyaddedtoBufferALasdescribedonpage16.Thissolutionshouldbepreparedfresh,andisstableat2–8°Cforupto48hours.UnusedportionsofcarrierRNAdissolvedinBufferAVEshouldbefrozeninaliquotsat–20°C.LyophilizedQIAGENProteasecanbestoredatroomtemperature(15–25°C)untilthekitexpirationdatewithoutaffectingperformance.QIAGENProteasereconstitutedinBufferAVEorProteaseResuspensionBufferisstableforupto1yearwhenstoredat2–8°C,butonlyuntilthekitexpirationdate.KeepingtheQIAGENProteasestocksolutionatroomtemperatureforprolongedperiodsoftimeshouldbeavoided.1063023_HB19.04.201014:49UhrSeite4Administrator高亮QIAampMinEluteVirusSpinHandbook04/20105QualityControlInaccordancewithQIAGEN’sISO-certifiedQualityManagementSystem,eachlotofQIAampMinEluteVirusSpinKitsistestedagainstpredeterminedspecificationstoensureconsistentproductquality.ProductUseLimitationsTheQIAampMinEluteVirusSpinKitisintendedformolecularbiologyapplications.Thisproductisnotintendedforthediagnosis,prevention,ortreatmentofadisease.Allduecareandattentionshouldbeexercisedinthehandlingoftheproducts.WerecommendallusersofQIAGENproductstoadheretotheNIHguidelinesthathavebeendevelopedforrecombinantDNAexperiments,ortootherapplicableguidelines.1063023_HB19.04.201014:49UhrSeite5QIAampMinEluteVirusSpinHandbook04/20106ProductWarrantyandSatisfactionGuaranteeQIAGENguaranteestheperformanceofallproductsinthemannerdescribedinourproductliterature.Thepurchasermustdeterminethesuitabilityoftheproductforitsparticularuse.Shouldanyproductfailtoperformsatisfactorilyduetoanyreasonotherthanmisuse,QIAGENwillreplaceitfreeofchargeorrefundthepurchaseprice.Wereservetherighttochange,alter,ormodifyanyproducttoenhanceitsperformanceanddesign.IfaQIAGENproductdoesnotmeetyourexpectations,simplycallyourlocalTechnicalServiceDepartmentordistributor.Wewillcredityouraccountorexchangetheproduct—asyouwish.SeparateconditionsapplytoQIAGENscientificinstruments,serviceproducts,andtoproductsshippedondryice.Pleaseinquireformoreinformation.AcopyofQIAGENtermsandconditionscanbeobtainedonrequest,andisalsoprovidedonthebackofourinvoices.Ifyouhavequestionsaboutproductspecificationsorperformance,pleasecallQIAGENTechnicalServicesoryourlocaldistributor(seebackcover).TechnicalAssistanceAtQIAGENweprideourselvesonthequalityandavailabilityofourtechnicalsupport.OurTechnicalServiceDepartmentsarestaffedbyexperiencedscientistswithextensivepracticalandtheoreticalexpertiseinmolecularbiologyandtheuseofQIAGENproducts.IfyouhaveanyquestionsorexperienceanydifficultiesregardingtheQIAampMinEluteVirusSpinKitorQIAGENproductsingeneral,pleasedonothesitatetocontactus.QIAGENcustomersareamajorsourceofinformationregardingadvancedorspecializedusesofourproducts.ThisinformationishelpfultootherscientistsaswellastotheresearchersatQIAGEN.Wethereforeencourageyoutocontactusifyouhaveanysuggestionsaboutproductperformanceornewapplicationsandtechniques.FortechnicalassistanceandmoreinformationpleasecalloneoftheQIAGENTechnicalServiceDepartmentsorlocaldistributors(seebackcover).1063023_HB19.04.201014:49UhrSeite6QIAampMinEluteVirusSpinHandbook04/20107SafetyInformationWhenworkingwithchemicals,alwayswearasuitablelabcoat,disposablegloves,andprotectivegoggles.Formoreinformation,pleaseconsulttheappropriatematerialsafetydatasheets(MSDSs).TheseareavailableonlineinconvenientandcompactPDFformatatwww.qiagen.com/ts/msds.aspwhereyoucanfind,view,andprinttheMSDSforeachQIAGENkitandkitcomponent.CAUTION:DONOTaddbleachoracidicsolutionsdirectlytowastecontainingBufferALorBufferAW1.BufferALandBufferAW1containguanidinehydrochloride,whichcanformhighlyreactivecompoundswhencombinedwithbleach.Ifliquidcontainingthesebuffersisspilt,cleanwithsuitablelaboratorydetergentandwater.Ifthespiltliquidcontainspotentiallyinfectiousagents,cleantheaffectedareafirstwithlaboratorydetergentandwater,andthenwith1%(v/v)sodiumhypochlorite.ThefollowingriskandsafetyphrasesapplytocomponentsoftheQIAampMinEluteVirusSpinKit:BufferALContainsguanidinehydrochloride:harmful,irritant.Riskandsafetyphrases:*R22-36/38,S13-26-36-46BufferAW1(concentrate)Containsguanidinehydrochloride:harmful,irritant.Riskandsafetyphrases:*R22-36/38,S13-26-36-46QIAGENProteaseContainssubtilisin:sensitizer,irritant.Riskandsafetyphrases:*R37/38-41-42,S22-24-26-36/37/39-4624-houremergencyinformationEmergencymedicalinformationinEnglish,French,andGermancanbeobtained24hoursadayfrom:PoisonInformationCenterMainz,GermanyTel:+49-6131-19240*R22:Harmfulifswallowed;R36/38:Irritatingtoeyesandskin;R37/38:Irritatingtorespiratorysystemandskin;R41:Riskofseriousdamagetoeyes;R42:Maycausesensitizationbyinhalation;S13:Keepawayfromfood,drinkandanimalfeedingstuffs;S22:Donotbreathedust;S24:Avoidcontactwithskin;S26:Incaseofcontactwitheyes,rinseimmediatelywithplentyofwaterandseekmedicaladvice;S36:Wearsuitableprotectiveclothing;S36/37/39:Wearsuitableprotectiveclothing,glovesandeye/faceprotection;S46:Ifswallowed,seekmedicaladviceimmediatelyandshowthecontainerorlabel.1063023_HB19.04.201014:49UhrSeite7QIAampMinEluteVirusSpinHandbook04/20108IntroductionTheQIAampMinEluteVirusSpinKituseswell-establishedtechnologyforsimultaneouspurificationofviralDNAandRNA.Thekitcombinestheselectivebindingpropertiesofasilica-basedmembranewithflexibleelutionvolumesofbetween20and150µl.Theprocedureissuitableforusewithplasma,serum,andothercell-freebodyfluids.Samplescanbeeitherfreshorfrozen,providedthattheyhavenotbeenfrozenandthawedmorethanonce(seepage15).ViralnucleicacidsareelutedinBufferAVE,readyforuseinamplificationreactionsorstorageat–20°C.Purifiednucleicacidsarefreeofproteins,nucleases,andotherimpurities.PrincipleandprocedureTheQIAampMinEluteVirusSpinprocedurecomprises4steps(lyse,bind,wash,elute)andiscarriedoutusingQIAampMinElutecolumnsinastandardmicrocentrifugeorfullyautomatedontheQIAcube™.Theprocedureisdesignedtoensurethatthereisnosample-to-samplecross-contaminationandallowssafehandlingofpotentiallyinfectioussamples.ThesimpleQIAampMinEluteSpinprocedure,whichishighlysuitedforsimultaneousprocessingofmultiplesamples,yieldspurenucleicacidinlessthan1hour.TheQIAampMinEluteVirusSpinKitcanbeusedforisolationofviralRNAandDNAfromabroadrangeofRNAandDNAviruses.However,performancecannotbeguaranteedforeveryvirusspeciesandmustbevalidatedbythecustomer.AutomatedviralnucleicpurificationontheQIAcubePurificationofviralnucleicacidsusingtheQIAampMinEluteVirusSpinKitcanbefullyautomatedontheQIAcube.TheinnovativeQIAcubeusesadvancedtechnologytoprocessQIAGENspincolumns,enablingseamlessintegrationofautomated,low-throughputsampleprepintoyourlaboratoryworkflow.TheQIAcubeperformsthesamestepsasthemanualprocedure(lyse,bind,wash,andelute)enablingyoutocontinueusingtheQIAampMinEluteVirusSpinKitforpurificationofhigh-qualityviralnucleicacids.TheQIAcubeispreinstalledwithprotocolsforpurificationofplasmidDNA,genomicDNA,RNA,viralnucleicacids,andproteins,plusDNAandRNAcleanup.Therangeofprotocolsavailableiscontinuallyexpanding,andadditionalQIAGENprotocolscanbedownloadedfreeofchargeatwww.qiagen.com/MyQIAcube.AdetailedprotocolforusingtheQIAampMinEluteVirusSpinKitontheQIAcubeispro-videdwiththeQIAcube.1063023_HB19.04.201014:49UhrSeite8Administrator高亮QIAampMinEluteVirusSpinHandbook04/20109Automatedviralnucleicacidpurification.ViralnucleicacidpurificationusingtheQIAampMinEluteVirusSpinKitcanbefullyautomatedontheQIAcube.SamplevolumesusingtheQIAampMinEluteVirusSpinKitEachQIAampMinElutecolumncanbindnucleicacidsthatarelongerthan200bases,butyielddependsonsamplevolumeandvirustiter.Thespinprocedureisoptimizedforusewithastartingvolumeof200µl.LysiswithQIAGENProteaseSamplesarelysedunderhighlydenaturingconditionsatelevatedtemperatures.LysisisperformedinthepresenceofQIAGENProteaseandBufferAL,whichtogetherensureinactivationofRNases.AdsorptiontotheQIAampMinElutemembraneBindingconditionsareadjustedbyaddingethanoltoallowoptimalbindingoftheviralRNAandDNAtothemembrane.LysatesarethentransferredontoaQIAampMinElutecolumnandviralnucleicacidsareadsorbedontothesilica-gelmembraneasthelysateisdrawnthroughbycentrifugation.SaltandpHconditionsensurethatproteinandothercontaminants,whichcaninhibitPCRandotherdownstreamenzymaticreactions,arenotretainedontheQIAampMinElutemembrane.QIAampMinElutecolumnsfitintomoststandardmicrocentrifugetubes.Duetothevolumeoffiltrate,2mlcollectiontubes(provided)arerequiredtosupporttheQIAampMinElutecolumnduringloadingandwashsteps.RemovalofresidualcontaminantsNucleicacidsremainboundtothemembrane,whilecontaminantsareefficientlywashedawayduring3washsteps.Inasinglestep,highlypureviralRNAandDNAareelutedinBufferAVE,equilibratedtoroomtemperature.1063023_HB19.04.201014:49UhrSeite9QIAampMinEluteVirusSpinHandbook04/201010ElutionofpurenucleicacidsElutionisperformedusingBufferAVE.TheQIAampMinElutecolumnsallowminimalelutionvolumesofonly20µl.Lowelutionvolumeleadstohighlyconcentratednucleicacideluates.Fordownstreamapplicationsthatrequiresmallstartingvolumes(e.g.,somePCRandRT-PCRassays)amoreconcentratedeluatemayincreaseassaysensitivity.Fordownstreamapplicationsthatrequirealargerstartingvolume,theelutionvolumecanbeincreasedupto150µl.However,anincreaseinelutionvolumewilldecreasetheconcentrationofnucleicacidsintheeluate.Theeluatevolumerecoveredcanbeupto5µllessthanthevolumeofelutionbufferappliedtothecolumn;forexample,anelutionbuffervolumeof20µlresultsin>15µlfinaleluate.Thevolumeofeluaterecovereddependsonthenatureofthesample.ElutedDNAcanbecollectedinstandard1.5mlmicrocentrifugetubes(notprovided).IfthepurifiedviralRNAandDNAistobestoredforupto24hours,storageat2–8°Cisrecommended.Forperiodsofstoragelongerthan24hours,storageat–20°Cisrecommended.YieldandsizeofviralnucleicacidsYieldsofviralnucleicacidisolatedfrombiologicalsamplesarenormallybelow1µgandarethereforedifficulttodeterminewithaspectrophotometer.Quantitativeamplificationmethodsarerecommendedfordeterminationofyields.WhenquantifyingnucleicacidsisolatedusingtheQIAampMinEluteVirusSpinprotocol,rememberthattherewillbeconsiderablymorecarrierRNAinthesamplethanviralRNA.Thesizedistributionofviralnucleicacidpurifiedwiththisprocedurecanbecheckedbyagarosegelelectrophoresisandhybridizationtoavirus-specificlabeledprobefollowedbyautoradiography(Sambrook,J.andRussell,D.W.[2001]MolecularCloning:ALab-oratoryManual,3rded.ColdSpringHarbor,NY:ColdSpringHarborLaboratoryPress).1063023_HB19.04.201014:49UhrSeite10QIAampMinEluteVirusSpinHandbook04/201011TheQIAampMinEluteVirusSpinProcedureSampleLyseBindPureviralnucleicacidsEluteWash(BufferAW2)Wash(ethanol)Dryspin(usenewcollectiontube)Wash(BufferAW1,recommended)FullyautomatableontheQIAcube1063023_HB19.04.201014:49UhrSeite11QIAampMinEluteVirusSpinHandbook04/201012CarrierRNACarrierRNAservestwopurposes.Firstly,itenhancesbindingofviralnucleicacidstotheQIAampmembrane,especiallyifthereareveryfewtargetmoleculesinthesample.Secondly,theadditionoflargeamountsofcarrierRNAreducesthechanceofviralRNAdegradationintherareeventthatRNasemoleculesescapedenaturationbythechaotropicsaltsanddetergentinBufferAL.IfcarrierRNAisnotaddedtoBufferALthismayleadtoreducedviralRNAorDNArecovery.TheamountoflyophilizedcarrierRNA(provided)issufficientforthevolumeofBufferALsuppliedwiththekit.TheconcentrationofcarrierRNAhasbeenadjustedsothattheQIAampMinEluteVirusSpinprotocolcanbeusedasagenericpurificationsystemcompatiblewithmanydifferentamplificationsystemsandissuitableforawiderangeofRNAandDNAviruses.Differentamplificationsystemsvaryinefficiencydependingonthetotalamountofnucleicacidpresentinthereaction.EluatesfromthiskitcontainbothviralnucleicacidsandcarrierRNA,andamountsofcarrierRNAwillgreatlyexceedamountsofviralnucleicacids.CalculationsofhowmucheluatetoaddtodownstreamamplificationsshouldthereforebebasedontheamountofcarrierRNAadded.Toobtainthehighestlevelsofsensitivityinamplificationreactions,itmaybenecessarytoadjusttheamountofcarrierRNAaddedtoBufferAL.AdditionofinternalcontrolsUsingtheQIAampMinEluteVirusSpinprotocolincombinationwithcommerciallyavailableamplificationsystemsmayrequiretheintroductionofaninternalcontrolintothepurificationprocedure.InternalcontrolRNAorDNAshouldbeaddedtogetherwiththecarrierRNAtothelysisbuffer.Foroptimalpurificationefficiency,internalcontrolmoleculesshouldbelongerthan200nucleotides,assmallermoleculesarenotefficientlyrecovered.Refertothemanufacturer’sinstructionsinordertodeterminetheoptimalconcentration.Usingaconcentrationotherthanthatrecommendedmayreduceamplificationefficiency.1063023_HB19.04.201014:49UhrSeite12QIAampMinEluteVirusSpinHandbook04/201013EquipmentandReagentstoBeSuppliedbyUserWhenworkingwithchemicals,alwayswearasuitablelabcoat,disposablegloves,andprotectivegoggles.Formoreinformation,consulttheappropriatematerialsafetydatasheets(MSDSs),availablefromtheproductsupplier.■Ethanol(96–100%)*■1.5mlmicrocentrifugetubes(e.g.,SarstedtSafety-Cap,cat.no.72.690orEppendorf®Safe-Lock,cat.no.0030120.086)†forelution■Pipettips(pipettipswithaerosolbarriersforpreventingcross-contaminationarerecommended)■Disposablegloves■Heatingblockforlysisofsamplesat56°C■Microcentrifuge(withrotorfor1.5mland2mltubes)■Vortexer■Forsamples<200µl:0.9%NaClsolution*Donotusedenaturedalcohol,whichcontainsothersubstancessuchasmethanolormethylethylketone.†Thisisnotacompletelistofsuppliersanddoesnotincludemanyimportantvendorsofbiologicalsupplies.1063023_HB19.04.201014:49UhrSeite13QIAampMinEluteVirusSpinHandbook04/201014ImportantNotesHandlingofQIAampMinElutecolumnsBecauseofthesensitivityofnucleicacidamplificationtechnologies,thefollowingprecautionsarenecessarywhenhandlingQIAampMinElutecolumnsinordertoavoidcross-contaminationbetweensamplepreparations:■CarefullyapplythesampleorsolutiontotheQIAampMinElutecolumn.PipetthesampleintotheQIAampMinElutecolumnwithoutwettingtherimofthecolumn.■Changepipettipsbetweenallliquidtransfers.Theuseofaerosol-barrierpipettipsisrecommended.■AvoidtouchingtheQIAampMinElutemembranewiththepipettip.■Afterallpulse-vortexingsteps,brieflycentrifugethemicrocentrifugetubestoremovedropsfromtheinsideofthelid.■Wearglovesthroughouttheentireprocedure.Incaseofcontactbetweenglovesandsample,changeglovesimmediately.CentrifugationQIAampMinElutecolumnswillfitintomoststandard1.5–2mlmicrocentrifugetubes.Additional2mlcollectiontubesareavailableseparately.CentrifugationofQIAampMinElutecolumnsisperformedat6000xg(8000rpm)inordertoreducecentrifugenoise.CentrifugingQIAampMinElutecolumnsatfullspeedwillnotaffectDNAorRNAyield.Centrifugationatlowerspeedsisalsoacceptable,providedthatnearlyallofeachsolutionistransferredthroughtheQIAampMinElutemembrane.Forthedryspinattheendofthewashingprocedureandforelution,centrifugationshouldbecarriedoutatfullspeed.Allcentrifugationstepsshouldbecarriedoutatroomtemperature.ProcessingQIAampMinElutecolumnsinamicrocentrifuge■ClosetheQIAampMinElutecolumnbeforeplacingitinthemicrocentrifuge.Centrifugeasdescribed.■RemovetheQIAampMinElutecolumnandcollectiontubefromthemicrocentrifuge.PlacetheQIAampMinElutecolumninanewcollectiontube.Discardthefiltrateandthecollectiontube.Pleasenotethatthefiltratemaycontainhazardouswasteandshouldbedisposedofappropriately.■OpenonlyoneQIAampMinElutecolumnatatime,andtakecaretoavoidgeneratingaerosols.1063023_HB19.04.201014:49UhrSeite14QIAampMinEluteVirusSpinHandbook04/201015■Forefficientparallelprocessingofmultiplesamples,werecommendfillingarackwithcollectiontubessothattheQIAampMinElutecolumnscanbetransferredaftercentrifugation.Usedcollectiontubescontainingthefiltratecanbediscarded,andthenewcollectiontubescontainingtheQIAampMinElutecolumnscanbeplaceddirectlyinthemicrocentrifuge.PreparationofRNAWhenpreparingviralRNA,workquicklyduringthemanualstepsoftheprocedure.IfyouhavenotpreviouslyworkedwithRNA,readtheAppendixonpage22beforestarting.BufferAVEisRNase-freeupondelivery.Itcontainssodiumazide,anantimicrobialagentthatpreventsgrowthofRNase-producingorganisms.However,asthisbufferdoesnotcontainanyRNaseinhibitors,itwillnotactivelyinhibitRNasesintroducedbyinappropriatehandling.ExtremecareshouldbetakentoavoidcontaminationwithRNaseswhenhandlingBufferAVE.SamplestorageAftercollectionandcentrifugation,plasmaorserumcanbestoredat2–8°Cforupto6hours.Forlong-termstorage,freezingat–20°Cor–80°Cinaliquotsisrecommended.Frozenplasmaorserumsamplesmustnotbethawedmorethanonce.Repeatedfreeze–thawingleadstodenaturationandprecipitationofproteins,resultinginreducedviraltitersandthereforereducedyieldsofviralnucleicacids.Inaddition,cryoprecipitatesformedduringfreeze–thawingwillclogtheQIAampMinElutemembrane.Ifcryoprecipitatesarevisible,theycanbepelletedbycentrifugationat6800xgfor3minutes.Theclearedsupernatantshouldberemovedandprocessedimmediatelywithoutdisturbingthepellet.Thisstepwillnotreduceviraltiters.PreparationofQIAGENProteaseThiskitprovidestwoalternativebuffersfordissolvingQIAGENProtease—BufferAVE(recommended)orProteaseResuspensionBuffer.DissolvingtheproteaseinBufferAVEprovidesagenericandefficientworkingsolutionforallstartingmaterials.Asanalternative,dissolvingQIAGENProteaseinProteaseResuspensionBufferprovidesefficientvirallysisformostsampletypes.Forsomestartingmaterials,suchasEDTAplasma,performanceisslightlyenhanced.However,ProteaseResuspensionBufferisnotcompatiblewithsamplesorinternalcontrolsthatcontainphosphate(e.g.,viraltransportmedium,cellculturesupernatants,orphosphate-bufferedsaline).Ifthesampleorinternalcontrolcontainsphosphate,itishighlyrecommendedtoresuspendQIAGENProteaseinBufferAVE.1063023_HB19.04.201014:49UhrSeite15QIAampMinEluteVirusSpinHandbook04/201016Add1.4mlofBufferAVEorProteaseResuspensionBuffertothevialoflyophilizedQIAGENProtease,andmixcarefullytoavoidfoaming.MakesurethattheQIAGENProteaseiscompletelydissolved.LabeltheresuspendedQIAGENProteasetoindicatewhichbufferwasusedforre