Northern杂交试剂盒说明书

Forlifescienceresearchonly.Notforuseindiagnosticsprocedures.FORINVITROUSEONLY.DIGNorthernStarterKitContentversion:August2010Fortranscription-labelingofRNAwithdigoxigeneinandSP6/T7/T3RNApolymerasesandchemiluminescentdetectionwithCDP-Star,ready-to-use.Cat.No.12039672910Kitfor10labelingreactionsanddetectionof10blotsof10ϫ10cm2StorethekitatϪ15toϪ25°C1.1.1PrefaceTableofContents1.Preface21.1TableofContents21.2Kitcontents32.Introduction52.1Productoverview53.Proceduresandrequiredmaterials83.1Beforeyoubegin83.2lowchart83.3DNAtemplatepreparation93.4DIG-RNAlabeling113.5Determinationoflabelingefficiency123.6GelsystemsuitableforDIGNorthernblots153.7RNAtransferandfixation163.8Hybridization173.9Immunologicaldetection193.10StrippingandreprobingofRNAblots214.FResults224.1Typicalresults225.5.4Appendix24Troubleshooting24Changestopreviousversion25References26OrderingInformation2621.2KitcontentsBottle/Cap1aLabelLabelingMixContentincludingfunction•40␮l•5×conc.labelingmixturecontainingoptimalconcentrationsofunlabelednucleotidesandDIG-11-UTP•Clearviscoussolution•ForefficientinvitrotranscriptionoflinearizedtemplateDNA•40␮l•5×conc.transcriptionbuffer•Clearsolution•ForefficientinvitrotranscriptionoflinearizedtemplateDNA•[20U/␮l]•SyntheziseRNAfromaDNAtemplate•20␮l•[20U/␮l]•SyntheziseRNAfromaDNAtemplate•[20U/␮l]•SyntheziseRNAfromaDNAtemplate1bTranscriptionBufferPolymeraseT7RNAPolymerasePolymerase35Anti-digoxigenin-AP,•60Fabfragments•[750U/ml]•Polyclonalsheepanti-digoxigenin,Fab-fragments,conjugatedtoalkalinephosphatase•ClearsolutionDNaseI,RNasefreeready-to-useActinRNAprobe,DIG-labeled•20␮l•[10U/␮l]•DegradesDNAtemplateaftertranscriptionreaction•Chemiluminescentsubstrateforalkalinephosphatase•Antisenseprobe,length588bases•[10ng/␮l]•StandardforthequantificationofDIG-labeledRNAandasahybridizationcontrol•ForthehybridizationoftheDIG-labeledRNAprobe••••2×100ml10×conc.Yellow,viscoussolutionForchemiluminescentdetectionprocedure6810GranulesBlockingsolutionAdditionalInadditiontothereagentslistedabove,youhavetoprepareseveralsolutions,equipmentandexclusivelywithDMPCorDEPCtreatedwater.Inthetableyouwillfindanoverviewreagentsrequiredabouttheequipmentwhichisneededforthedifferentprocedures.Detailedinformationisgiveninfrontofeachprocedure.Preparationusing3.3.1standardmethodor3.3.1RT-PCRandPCRThermocycler•Phenol/Chloroform•ExpandHighFidelityPCRSystem(incl.buffer)*•Primer(sense,antisense)•Nucleotides(dATP,dCTP,dGTP,dTTP,each10mM)2•EDTA,0.2M,pH8.0•DIGWashandBlockBufferSet*or•Washingbuffer•Maleicacidbuffer•Detectionbuffer•10×MOPS,pH7,0(withNaOH)•Loadingbuffer•Gelsolution•Runningbuffer•20×SSCor•2×SSC•2×SSC,0.1%SDS•0.1×SSC,0.1%SDSDIG-RNAlabelinglabelingefficiency••••Ice/waterUV-transilluminatororUV-crosslinkerorOven(120°Cor80°C)forNorthernblots3.6.1Formaldehydegels••••andfixation•••••••••detectionGelequipmentHeatablewater-bath(65°C)orHeatingblock(65°C)IceWhatmann3MMpaperUV-transilluminatororUV-crosslinkerorOven(120°Cor80°C)Ice/waterWater-bathShakingwater-bathorhybridizationovenHybridizationbags*or•Temperatureresistant,sealableplasticorglassboxes,petridishesorrollerbottlesDonotuseopentraysorboxeswithDIGEasyHyb.•DevelopmentfoldersreprobingofRNAblots•Hybridizationbags*or•Washingbuffer•Maleicacidbuffer•Detectionbuffer•Strippingbuffer•2×SSC12039672910DIGNorthernStarterKit2.2.1IntroductionProductoverviewTheDIGNorthernStarterKitgeneratesDIGlabeled,singlestrandedRNAprobesofdefinedlengthbyinvitrotranscriptionoftemplateDNAinthepresenceofdigoxigenin-UTP,usingSP6,T7orT3RNApolymerases.StageRNAlabelingDescriptionDIG-labeledRNAprobesaregeneratedaccordingtotheinvitrotranscriptionlabelingtechnique.ThelabelingmixcontainsoptimalconcentrationsofnucleotidesandDIG-11-UTPandaspeciallydevelopedtranscriptionbufferarecombinedwiththelinearizedDNAtemplateandtheappropriateRNApolymerase.DIG-labeledRNAprobesareusedforhybridizationtomembraneblottednucleicacidsaccordingtostandardmethods.TestprincipleHybridizationdetectionFabfragmentsandarethenvisualizedwiththechemiluminescencesubstrateCDP-Star,ready-to-use.EnzymaticdephosphorylationofCDP-Star,ready-to-usebyalkalinephosphataseleadstoalightemissionatamaximumwavelengthof465nm(seefig.1)whichisrecordedwiththeimagingdeviceoronX-rayfilms.Exposuretimesareintherangeof5to30min,only.Fig.1:ReactionofCDP-StarApplicationSamplematerialNorthernblotsonnylonmembranes•Linearizedplasmid,includingtheappropriateRNApolymerasepromotersequence(SP6,T3,T7).1000bp.ToavoidRNasecontaminationtheDNAmustbephenolized.•SpeciallypreparedPCRproduct(seechapter3.3.2)ThistableliststhereactiontimeofthesinglestepsStepRNAlabelingHybridizationImmunologicaldetectionChemiluminescentsignaldetectionReactiontime1h20min6horovernight1h40min5-30minAssaytimeNumberoftests1kitissufficientfor•10labelingreactionsof1␮gDNA,yieldingapprox.20␮glabeledRNA,eachanddetectionof•10blotsof10×10cm2.6Kitstorage/stabilityTheunopenedkitisstableatϪ15toϪ25°Cuntiltheexpirationdateprintedonthelabel.Onceopened,pleaserefertothefollowingtableforappropriatestorageKitcomponentLabelingMixandTranscriptionBufferRNApolymerasesAnti-digoxigenin-AP,Fabfragmentsvial1a1b2-45Storage/StabilityStableatϪ15toϪ25°C:Repeatedfreezingandthawingshouldbealiquotthesolutionsandtostorein2-3portions.StableatϪ15toϪ25°CStableat+2to+8°C:Donotfreeze!:Storeprotectedfromlight!•Oncedissolved,thesolutionisstablefor1monthwhenkeptsterile.Stableat+2to+8°CBlockingsolution(10xconcentrated)10SensitivityandspecificityAdvantagesRaremRNAscanbedetectedin0.1␮goftotalRNAThistabledescribesbenefitsandfeaturesofthekit:BenefitAccurateandfastFeatureTheuseoftheapplicationtestedandoptimizedkitcomponentsminimizesthehands-on-timerequiredtolabelRNAprobesandincreasesefficiencyandreproducibility.ethanolforatleastoneyear.Werecommendtofreezetheprobeinaliquots,andavoidrepeatedfreeze/thaw12039672910DIGNorthernStarterKit3.ProceduresandrequiredmaterialsBeforeyoubeginRNase-freeconditions2allsolutions•Autoclavesolutions•Tween20shouldbeaddedtopreviouslysterilizedsolutionsZAP(SigmaR-2020)orbakeglasstrays8hoursat200°Cbeforeuse•Wearpowder-freegloves•HandlemembraneonlyontheedgeswithcleanforcepsMembranehandlingrequirements3.2FlowchartDNAtemplatepreparationDIG-RNAlabeling↓Determinationoflabelingefficiency↓SuitablegelsystemsforDIGNorthernblots↓RNAblottingandfixation↓Hybridization↓Immunologicaldetection↓StrippingandreprobingofRNAblots12039672910DIGNorthernStarterKit3.3.1DNATemplatePreparationStandardmethod•PlasmidDNAshouldbepurifiedusingtheHighPurePlasmidIsolationKit*.•DNAtemplatemustbelinearizedatarestrictionsitedownstreamoftheclonedinsert.Thesequencetobetranscribedshouldbe200to1000bpinlength.•Toavoidtranscriptionofundesirablesequences,usearestrictionenzymethatleaves5’overhangorbluntends.•Afterrestrictiondigest,purifytheDNAbyphenol/chloroformextraction,followedbyethanolprecipitation.•DissolvetemplateDNAin10mMTris,0,1mMEDTA,pH8,00.1mMEDTAintheTEbufferfordissolvingtheDNA.Generalinformation3.3.2PreparingDNATemplatefromtotalRNAusingRT-PCRandPCRThefollowingtabledescribesanalternativemethodforgeneratingtemplatesforinvitrotranscriptionlabelingofRNAwithDIGwithoutcloning.sequencesexperimentshaveshownthatSP6Polymerasecanonlyinitiateefficienttranscriptionifthepromotersequenceslieswithinaplasmidenvironment(3).or5'TAATACGACTCACTATAGGA/X-mer5'AATTAACCCTCACTAAAGGG/X-merT3RNApolymeraseAdditional•Thermocyclerequipmentand•ExpandHighFidelityPCRSystem,includingbufferreagentsrequired•Primer(sense,antisense)•Nucleotides(dATP,dCTP,dGTP,dTTP,each10mM)ProcedureFindinthefollowingtabletherecommendedprotocolusingtheExpandHighFidelityPCRSystem*.ice)inthefollowingorder:Reagentsteriledoubledist.water,DMPCorDEPC-treatedcontainingMgCl2(suppliedwiththeenzyme)10mMdATP,dCTP,dGTP,dTTPPrimer1(sense)Primer2(anti-sense)ExpandHighFidelitycDNAFinalvolume•Mixgentlyandcentrifugebriefly.2Cycle30×asfollows:Volumevariableeach1␮lvariable0.75␮l2␮l50␮lFinalconc.-2(1×)0.2mMvariable300nM12039672910DIGNorthernStarterKit3.4DIG-RNALabelingRNAislabeledinaninvitrotranscriptionreactionwithdigoxigenin-11-UTPusingaLabelingmixtureandanoptimizedTranscriptionBuffer.IntroductionAdditional•Waterbathfor42°Cand37°Corheatingblockequipmentand•Ice/waterreagentsrequiredThistablelistscomposition,storageanduseoftheadditionallyrequiredreagents.SolutionEDTACompositiondoubledistilledwaterStorage/StabilitystableUsesolutionsStoppingthereactionProcedureThisprocedureisdesignedfor1μgofDNAtemplate.Largeramountscanbelabeledbyscalingupofallcomponentsandvolumes.Step1ActionAdd1␮glinearizedplasmidDNAor4␮lPCRproduct(100-200ng)andsterileRNase-free,DMPCorDEPCtreated,doubledist.watertoafinalvolumeof10␮ltoasterilizedreactionvial.•Mixandcentrifugebriefly.•Incubatefor1hat42°C.•Incubatefor15minat37°C.Labeling•Inthestandardreactionwith1␮gDNAperassay67%ofthenucleotidesareincor-efficiencyandsizeporatedintoapprox.20␮gofnewlysynthesizedDIG-labeledRNAwithin1h.oflabeledRNA•ThesizeofthelabeledRNAisintherangeof200-100012039672910DIGNorthernStarterKit3.5DeterminationoflabelingefficiencyDeterminationoftheyieldofDIG-labeledRNAisveryimportantforoptimalandreproduciblehybridizationresults.Toohighprobeconcentrationsinthehybridizationstepcausesbackground,whiletoolowconcentrationsleadstoweaksignals.ThepreferentialmethodfordeterminationoflabelingefficiencyofprobesisthedirectdetectionmethodincomparisontothecontrolRNA(vial8).Stage1Description•AseriesofdilutionsofDIG-labeledRNAisappliedtoasmallstripofnylonmembrane,positivelycharged*.•PartofthenylonmembraneispreloadedwithdefineddilutionsofcontrolRNA,usedasstandards.anti-digoxigenin-APandCDP-Starready-to-use.•TheintensitiesofthedilutionseriesofDIG-labeledRNAandcontrolRNAarecomparedbyexposuretoimagingdeviceorX-rayfilm.IntroductionTestprincipleAdditionalequipmentrequired••••Nylonmembranes,positivelycharged*UV-transilluminatororUV-crosslinkerorOven(120°Cor80°C)PreparationofTheWashingbuffer,Maleicacidbuffer,Blockingsolution,andDetectionbufferarealsoavailableinaready-to-useformintheDIGWashandBlockBufferSetadditionalsolutionsrequiredDNase-andRNase-free*.ThesesolutionsarealsousedinthedetectionprocedureofChapter3.9andcanbepreparedinlargeramounts.SolutionRNAdilu-tionbufferbufferComposition/PreparationMixDMPCtreateddoubledist.water:20×SSC:Formaldehyd(37%)intheratio5:3:20.3%(v/v)Tween20Stabilitypreparefresh+25°C,stable+15to+25°C,stable+15to+25°C,stableUseDilutionofRNAunspecificboundantibodyDilutionofBlock-ingsolutionAdjustmentofpHMaleicacid0.1MMaleicacid,0.15MNaCl;adjustbufferwithNaOH(solid)topH7.5Detectionbuffer0.1MTris-HCl,0.1MNaCl,pH9.5(20°C)12039672910DIGNorthernStarterKitPreparationofkitPleaserefertothefollowingtableforthepreparationoftheworkingsolutions.workingsolutionsBlockingsolutionAntibodysolutionStabilityPreparea1×workingsolutionbydilu-Alwayspre-Blockingofun-parefreshspecificbindingtingthe10×Blockingsolution(bottle10)1:10inMaleicacidbuffer.sitesonthemem-braneCentrifugeanti-digoxigenin-AP(vial5)+2to+8°CBindingtotheDIG-labelfor5minat10000rpmintheoriginalfor2hvialpriortoeachuse,andpipetthenecessaryamountcarefullyfromthesurface.Diluteanti-digoxigenin-AP1:10,000(75mU/ml)inBlockingsolu-tion.DilutionseriesPrepareadilutionseriesofyourlabeledprobeandyourcontrolRNAasdescribedinthetable,wheretube1iseitheradilutionofyourlabelingreactionto10ng/␮l(expectedyieldofastandardlabelingreactionis20␮goflabeledRNA)orthecontrolRNA(vial8)whichalsohastheconcentrationof10ng/␮l.1(␮l)-tube#dilutedlabelingreactionorvial8DilutionBuf-fer(␮l)--concentration10ng/␮12039672910DIGNorthernStarterKitActionApply1␮lspotsoftubes3-10fromyourlabeledprobesandthelabeledcontroltothenylonmembrane.for30minat120°C•Incubateundershakingfor2minat15-25°C.(orhybridizationbag)andapplyCDP-Starapprox.4drops(fromthedrop-perbottle7)tothemembrane.•Immediatelycoverthemembranewiththesecondsheetofthefoldertospreadthesubstrateevenlyandwithoutairbubblesoverthemembrane.Incubatefor5minat+15to+25°C.theresultComparetheintensityofthespotsoutofyourlabelingreactiontothecontrolandcalculatetheamountofDIG-labeledRNA.143.6GelSystemsuitableforDIGNorthernBlots•StandardprotocolsforgelelectrophoresiscanbeusedasdescribedinSambrooketal.[2].•Gelslackingethidiumbromidearepreferentialused,becauseethidiumbromidecancauseunevenbackgroundproblems.TargetamountsIfyouareusingtotalRNAmRNAThenloadmaximally1µgperlaneload100ngperlane3.6.1FormaldehydeGelSolutionsrequiredThefollowingbuffersarerequiredforrunningaformaldehydegel:Solution10×MOPS,pH7.0(withNaOH)(alwayspreparefresh)Composition200mMMOPS50mMNaAc20mMEDTA83␮lof37%Formaldehyde50␮l10×MOPS50␮l100%Glycerol10␮l2.5%Bromphenolblue57␮lDEPC/DMPCtreatedwater1.8gAgarose141.9ml1×MOPS8.1ml37%Formaldehyde1×MOPSGelwithFormaldehyd(2%)RunningbufferPreparationofsamplesFindthefollowingtablethepreparationofthesamplesStep12345ActionAdd20µl(or2-3volume)ofLoadingbuffertotheRNAprobe.DenaturetheRNAprobe/Loadingbuffermixat65°Cfor10min.ChilltheRNAprobe/Loadingbuffermixonicefor1min.Runthegelwit3-4V/cminRNasefreegelboxesforatleast2h(preferablyover-night)untiltheRNAsarewellseparated..ToaccessthequalityofthetargetRNAafterelectrophoresis,stainthegelbrieflyin0.25-0.5µg/mlethidiumbromideandexaminethegelunderUVlight.3.7RNAtransferandfixationTransfermethods•Whenusingformaldehydegels,pleaserinsegelspriortoblottingfor2×15mininandmembranes20×SSC•AllcommontypesofRNAtransfermethodsaresuitableforsubsequentDIGhybridization[7]•Bestresultsareobtainedwhengelsareblottedbycapillarytransferwith20×SSCovernightoratleastfor6hours•Useonlynylonmembranes,positivelychargedWedonotrecommendtousealkalitransfer(e.g.,in0.4MNaOH)forRNAandDIG-labeledmolecularweightmarkers*.FixationprocedureFixtheRNAtothemembranebyanyofthefollowingprocedures.:UsesterileandRNase-freesolutionsandequipmentIFyouwantto...UV-crosslinkingTHEN...•placethemembraneonWhatman3MM-papersoakedwith2×SSC.•UV-crosslinkthewetmembranewithoutpriorwashing.•aftertheUV-crosslinking,rinsethemembranebrieflyindoubledistilledwaterandallowtoair-dry.•bakethenylonmembraneat120°Cfor30minoraccordingtothemanufacturer´sinstructions.•rinsemembrane2×brieflyin2×SSC•bakeat80°Cfor2hbakeat80°CStorageofthemembraneFindinthefollowingtablethestorageconditionsforthemembrane.IF...youwanttogoahead.•ThedegreeofhomologyoftheprobetothetargetRNA(see”hybridizationtemperature”)isanimportantfactorfordeterminingtheappropriateconditionsforhybridization.•Stringencyisinfluencedbytemperature:hightemperatureincreasesstringencyofhybridization,lowtemperaturedecreasesstringency.•ForRNA:RNAhybridizazionswithDIGEasyHyb,werecommendgenerallyahybridizationtemperatureof68°C.ThetemperaturemayhavetobeadjusteddependingontheGCcontentandhomologyofprobetotarget.•••••••Nylonmembranes,positivelycharged*Ice-waterWater-bathShakingwater-bathorHybridizationovenHybridizationbags*orTemperatureresistantplasticorglassboxes,petridishes,rollerbottlesorsealableplasticbags.Factorswhichinfluencestrin-gencyAdditionalequipmentrequiredAdditional•20×SSCsolutionsrequired•10%SDSPreparationofkitPleaserefertothefollowingtabletofindthepreparationprotocolforthekitworkingworkingsolutionsolution.:Usesterile,RNase-freesolutionsandequipment!SolutionEasyHybGranulesComposition/Preparationadding64mlsteriledoubledistilled,DEPCorDMPCtreatedwaterintwoportionstotheplasticbottle,dissolvebystirringat37°C.dissolvedunderRNase-freeconditions.Storage/Stability+15to+25°Cfor1monthUseprehybridizationandhybridizationsolutionProcedurePleaserefertothefollowingtable.100%homologyoftheprobetothetargetsequence),theDIGEasyHybworkingsolu-tionusedforprehybridizationhastobeprewarmedto68°C.Step1Action•PrewarmanappropriatevolumeofDIGEasyHyb(10-15ml/100cmfilter)tohybridizationtemperature(68°C).•PrehybridizemembranewithDIGEasyHybfor30minwithgentleagita-tioninanappropriatecontainer.Membranesshouldmovefreely,especiallyifyouuseseveralmembranesinthesameprehybridizationsolution.ice/water.RNAisdegradedbyalkali-solutions!AdddenaturedDIG-labeledRNAprobe(100ng/ml)toprewarmedDIGEasyHyb(3.5ml/100cm2membrane)andmixwellbutavoidfoaming(bubblesmayleadtobackground).tomembrane.•Incubatefor6horO/Nat68°Cwithgentleagitation.3StorageofhybridizationsolutionInprinciple,DIGEasyHybcontainingDIG-labeledprobecanbestoredatϪ15toϪ25°Candbereusedwhenfreshlydenaturedat65°Cfor10minbeforeuse.WedonotrecommendreusageofDIGEasyHybcontainingRNAprobes,becausesuccessfulreusedependsoninherentprobestabilityandRNase-freeworkingconditions.Pleaserefertothefollowingtabletofindtheprocedureforstringencywashes.2agitation.Wash2×15minutesin0.1×SSC,0.1%SDS(prewarmedtowashtemperature)at68°Cunderconstantagitation.StringencywashesThestringencyofthefinalwashmustbedeterminedempirically.Dependingonhomologousprobeswilloftenrequire0.1xwashsolution.3.9Immunologicaldetection•••••••••Workundersterile,RNase-freeconditionsIfthemembraneistobereprobed,donotallowthemembranetodryatanytime.Usesufficientvolumeofallsolutions.Makesurethatmembranesdonotsticktotraysduringdetection.Avoidthatmembranessticktogether.Shakemembranesduringthewholeprocedure.Whenusinglaboratorytraysforthedetectionprocedure,theyshouldberigorouslycleanedbeforeuse.Workundercleanconditionswhenhandlingthechemiluminescentsubstratesolutionandavoidphosphatasecontamination.Forchemiluminescentdetection,membranesmustalsobesealedinplasticbagsordevelopmentfoldersDonotuse”SaranWrap”.GeneralinformationAdditionalreagentsrequiredTheWashingbuffer,Maleicacidbuffer,Blockingsolution,andDetectionbufferarealsoavailableinaready-to-useformintheDIGWashandBlockBufferSet,DNaseandRNasefree*.WashingbufferferPreparationofkitTheantibodysolutionshouldhavebeenpreparedfortheSemi-quantitativeworkingsolutionsdeterminationoflabeling(section3.4)otherwisefindinthefollowingtablethedescriptionforthepreparationoftheantibodyworkingsolution.SolutionComposition/PreparationStorage/StabilityAlwayspreparefresh2hat+2to+8°CUseBlockingofunspecificbindingsitesonthemembraneBindingtotheDIG-labeledprobeBlockingsolutionPreparea1×workingsolutionbydiluting10×Blockingsolution(bottle10)1:10withMaleicacidbuffer.AntibodysolutionCentrifugeanti-digoxigenin-AP(vial5)for5minat10000rpmintheoriginalvialpriortoeachuse,andpipetthenecessaryamountcarefullyfromthesurface.Diluteanti-digoxigenin-AP1:10000(75mU/ml)inBlockingsolution.3456Washingbuffer.Incubatefor30minin100mlBlockingsolution.Incubatefor30minin50mlAntibodysolution.Wash2×15minin100mlWashingbuffer.Equilibrate2-5minin100mlDetectionbuffer.•Placemembrane(withRNAsidefacingup)onadevelopmentfolder(orhybridizationbag)andquicklyapplyapprox.1ml.CDP-Star,ready-to-usesolutionoutofthedropperbottle(bottle7)untilthemembraneisevenlysoaked.•Immediatelycoverthemembranewiththesecondsheetofthefoldertospreadthesubstrateevenlyandwithoutairbubblesoverthemembrane.•Incubatefor5minat+15to+25°C.aroundthedampmembrane.:Dryingofthemembraneduringexposurewillresultindarkbackground.3.10StrippingandreprobingofRNAblotsOnlymembranes,whichdidnotdryatanytimeduringthehybridizationanddetectionprocedure,canbestripped.•Hybridizationbags•Water-bathBeforeyoubeginAdditionalequipmentrequiredAdditionalInthefollowingtablefindtheadditionalreagentsrequiredforthestrippingofreagentsrequiredmembranes.:Usesterile,RNasefreesolutions.H2OSterileDMPCorDEPCtreateddoubledistilledwaterStability+15to+25°C,stablealwayspre-parefreshWashingthemembraneRemovingDIG-labeledprobeStrippingbuffer50%Formamide50mMTris/HCl7,55%SDS20×SSCstocksolution3MNaCl300mMsodiumcitrate,pH7.0(Cat.No.1666681)+15to+25°C,Stocksolutionstableforthepreparationof2×SSCstorageofmembrane1:10withdoubledistilled,DEPCstableorDMDC-tratedwater.ProcedureThistabledescribeshowtoperformthestrippingprocedure.Step1ActionRinsemembranethoroughlyinsterileDMPCorDEPCtreateddoubledistilledwater.DIG-labeledprobe.sealedbag.Storageofstripped4.emplateDIG-labeledTSHreceptor(100ng/ml)Lane1:0.1␮gThyroidRNALane2:0.5␮gThyroidRNA12Fig.3Differenttissues,normalizedtoActin,hybridizedwithTSH-receptorprobe(100ng/ml)inaNorthernBlot.ProbeDIG-labeledTSHreceptorprobe(100ng/ml)andDIG-labeledActinprobe(100ng/ml).12345675.AppendixTroubleshootingTroubleshootingThistabledescribesvarioustroubleshootingparametersforDIG-labelinganddetectiontableProblemLowsensitivityPossiblecauseInefficientprobelabelingRecommendation•Checklabelingefficiency(section3.5).•PurifytemplateDNAbyphenolizationandethanolprecipitation•Makesurethatthetemplatewaslinearizedbeforelabeling.•Donotstoretemplateinbufferscontainingmorethan0.1mMEDTA.•ChecktheamountandqualityoftargetRNA100ng/ml.•Prolonghybridizationtimetoovernight.Determineoptimalprobeconcentrationasdescribedinsection3.5,donotusemorethan100ng/ml.time.Makesurethattheprobedoesnotcontaincross-hybridizingvectorsequences.Makesurethatthemembranewassoakedinsufficientprehybridizationsolution.background:usenylonmembranesfromRocheMolecularBiochemicals,testedfortheDIG-System.•Checktemperatureofstringencywashes,prewarmwashsolutiontocorrecttempera-ture.•Use0.1xSSCforhighstringencywashconcentrationinthehybridizationHighbackgroundConcentrationoflabeledprobeistoohighbraneProbesequenceInefficientprehybridizationnylonmembraneInefficientstringencywashes12039672910DIGNorthernStarterKitProblemSmearinlanesPossiblecauseTargetconcentrationtoohighRecommendationDonotusemorethan1␮goftotalor100ngofmRNAperlane.TheDIGSystemismoresensitivethanradioactivityandhigherRNAcon-centrationsresultindetectionofdegradationproducts.RNasefreeequipmentforthewholedetctionprocedure.•Donotallowthemembranetodryduringdetectionprocedure.•Handlemembranesonlywithpowder-freegloves.•Usesufficientvolumesofallsolutions.•Makesurethatmembranesdonotsticktotraysduringdetec-tion.Avoidthatmembranessticktogether.•Shakemembranesduringthewholedetectionprocedure.•Whenusinglaboratorytraysforthedetectionprocedure,theyshouldberigorouslycleanedbeforeuse.Anti-DIG-APbind-ingandchemiluminescentdevelopmentshouldbedoneinseparatetrays.•Workundercleanconditionswhenhandlingthechemilumi-nescentsubstratesolution,andavoidphosphatasecontaimina-tion.•Forchemiluminescentdetec-tionmembranesshouldalsobesealedinplasticbags.•Donotuse“SaranWrap”.5.2ChangestopreviousversionEditorialcorrections.Updatetrademarkinformation.Deletedlicensedisclaimer.RegulatoryDisclaimerTrademarksDIGEASYHYB,EXPAND,andHIGHPUREaretrademarksofRoche.CD