HiScribe™ T7 High Yield RNA Synthesis Kit - NEB

The HiScribe T7 High Yield RNA Synthesis Kit is an extremely flexible system for in vitro transcription of RNA using T7 RNA Polymerase. The kit allows for ... Home RNAReagents Products HiScribe™T7HighYieldRNASynthesisKit HiScribe™T7HighYieldRNASynthesisKit Upto180μgofRNAperreactionfrom1μgofcontroltemplate EnablesfullsubstitutionofNTPsforlabelingandincorporationofmodifiedbases LinearizedcontroltemplateincludedforverificationofRNAsynthesis KitComponents Manuals FeaturedVideo + AvoidingRNaseContamination Catalog# Concentration Size E2040S NotApplicable 50reactions Tofindouthowtoorderthisproductfromyourcurrentlocation,clickthebuttonbelow: InternationalCustomer&OrderSupport Pleaseenteraquantityforatleastonesize ProductInformation TheHiScribeT7HighYieldRNASynthesisKitisanextremelyflexiblesystemforinvitrotranscriptionofRNAusingT7RNAPolymerase.ThekitallowsforsynthesismanykindsofRNAincludinginternallylabeledandco-transcriptionallycappedtranscripts. RNAsynthesizedfromthekitissuitableformanyapplicationsincludingRNAstructureandfunctionstudies,ribozymebiochemistry,probesforRNaseprotectionassaysandhybridizationbasedblots,anti-senseRNAandRNAiexperiments,microarrayanalysis,microinjection,andinvitrotranslationandRNAvaccines. Thekitcontainssufficientreagentsfor50reactionsof20μleach.Eachstandardreactionyieldsupto180μgofRNAfrom1μgcontroltemplate.Eachkitcanyieldupto9mgRNA.For32Plabeling,thekitcontainsenoughreagentsfor100 reactionsof20μleach.  MaterialsNotIncluded: DNATemplate:TheDNAtemplatemustbelinearandcontaintheT7 RNAPolymerasepromoterwithcorrectorientationinrelationtotargetsequencetobetranscribed.  3′-O-Me-m7G(5′)ppp(5′)GRNACapStructureAnalog(NEB#S1411) m7G(5′)ppp(5′)GRNACapStructureAnalog(NEB#S1404) m7G(5′)ppp(5′)ARNACapStructureAnalog(NEB#S1405) G(5′)ppp(5′)ARNACapStructureAnalog(NEB#S1406) G(5′)ppp(5′)GRNACapStructureAnalog(NEB#S1407) Modified-NTP:Biotin-,Fluorescein-,Digoxigenin-,orAminoallyl-NTP  Labeling:[α-32P]labeledribonucleotide(800-6,000Ci/mmol)  General:37°CincubatororPCRmachine,nuclease-freewater  DNaseI:DNaseI(RNase-free)(NEB#M0303)  Purification:Buffer-orwater-saturatedphenol/chloroform,ethanoland3Msodiumacetate,pH5.2,spincolumns  GelAnalysis:Gelsandrunningbuffers,gelapparatus,powersupply Figure1.TranscriptionbyT7RNAPolymerase Figure2.TimecourseofstandardRNAsynthesisfromthreeDNAtemplates Reactionswereincubatedat37°CinaPCRmachine.TranscriptswerepurifiedbyspincolumnsandquantifiedonNanoDrop™Spectrophotometer. Figure3.EffectoftemplateamountonRNAyield Standardreactionswereincubatedat37°CinaPCRmachinefor2hours.TranscriptswerepurifiedbyspincolumnsandquantifiedonNanoDrop™Spectrophotometer. Figure4:ImprovedRNAyieldandintegrityfromextendeddurationtranscriptionreactions reactionswereassembled,induplicate,accordingtothemanufacturers’suggestedprotocolsusing3ngofdsDNAtemplateencodinga1.8kbRNA,andincubatedat37°Cfor16,24and40hours.Ateachtimepoint,thecorrespondingtubesweretransferredto-20°Ctostopthereaction.Transcriptionreactionswerecolumnpurifiedafterthelasttimepoint. (A)Transcriptyield–Aftercolumnpurification,RNAconcentrationwasmeasuredusingaNanoDropspectrophotometerandtotalRNAyieldwascalculated.ThesedatademonstratethatasubstantiallyhigheryieldofRNAwassynthesizedusingtheHiScribeT7HighYieldRNASynthesisKitascomparedtothecompetitor’skit. (B)Transcriptintegrity–150ngofcolumnpurifiedRNAwasruna1.2%denaturingagarosegel,stainedwithethidiumbromideandvisualizedbyUVfluorescence.ThedatademonstrategreatlyimprovedtranscriptintegrityafterextendeddurationRNAsynthesisreactionsusingtheHiScribeT7HighYieldRNASynthesisKitascomparedtothecompetitor’skit. Thisproductisrelatedtothefollowingcategories: RNASynthesisProducts Thisproductcanbeusedinthefollowingapplications: RNALabeling, InvitroTranscriptionforRNASynthesis KitComponents KitComponentsThefollowingreagentsaresuppliedwiththisproduct:NEB#ComponentNameComponent#Storedat(°C)AmountConcentrationE2040S  -20   CTP N0454AVIAL-201x0.1ml100mM FLucControlTemplateN0426AVIAL-201x0.01ml0.5µg/µl UTP N0453AVIAL-201x0.1ml100mM ATPN0451AVIAL-201x0.1ml100mM T7RNAPolymeraseMixM0255AVIAL-201x0.1mlNotApplicable GTP N0452AVIAL-201x0.1ml100mM 10XT7ReactionBufferB2041AVIAL-201x0.1mlNotApplicable Properties&Usage RelatedProducts CompanionProductsRNALoadingDye,(2X)RNaseInhibitor,HumanPlacentaRNaseInhibitor,MurineDNaseI(RNase-free)Q5®HotStartHigh-FidelityDNAPolymerasessRNALadderLowRangessRNALadder3´-0-Me-m7G(5')ppp(5')GRNACapStructureAnalogm7G(5')ppp(5')ARNACapStructureAnalogG(5')ppp(5')ARNACapStructureAnalogG(5')ppp(5')GRNACapStructureAnalogm7G(5')ppp(5')GRNACapStructureAnalogVacciniaCappingSystemmRNACap2OMethyltransferaseE.coliPoly(A)PolymeraseRibonucleotideSolutionMixRibonucleotideSolutionSet Protocols,Manuals&Usage Protocols DNATemplatePreparation(E2040)RNASynthesiswithModifiedNucleotides(E2040)PurificationofSynthesizedRNA(E2040)StandardRNASynthesis(E2040)CappedRNASynthesis(E2040)HighSpecificActivityRadiolabeledRNAProbeSynthesis(E2040)EvaluationofReactionProducts(E2040)Poly(A)TailingofRNAusingE.coliPoly(A)Polymerase(NEB#M0276)ProtocolforCo-transcriptionalcappingusingCleanCap®ReagentAGfromTriLinkandHiScribe™T7HighYieldRNASynthesisKitfromNewEnglandBiolabs® Manuals TheProductManualincludesdetailsforhowtousetheproduct,aswellasdetailsofitsformulationandqualitycontrols.manualE2040 FAQs&Troubleshooting FAQs CanIusetheMonarchRNACleanupKitstocleanupmyinvitrotranscription(IVT)reaction?HowcanIimproveonalowyieldofRNAfromthetranscriptionreaction?Aremodifiednucleotidesincludedinthekit? Troubleshooting ControlReaction  TheFLuccontroltemplateDNAisalinearizedplasmidcontainingthefireflyluciferasegeneunderthetranscriptionalcontrolofT7promoter.Thesizeoftherunofftranscriptis1.8kb.Thecontrolreactionshouldyield≥150μgRNAtranscriptin2hours. Ifthecontrolreactionisnotworking,theremaybetechnicalproblemsduringreactionsetup.Repeatthereactionbyfollowingtheprotocolcarefully;takeanyprecautiontoavoidRNasecontamination.ContactNEBfortechnicalassistance. Thecontrolplasmidsequencecanbefoundhere.TheFLuccontroltemplateisgeneratedbylinearizingtheplasmidwithStuI. LowYieldofFull-lengthRNA Ifthetranscriptionreactionwithyourtemplategeneratesfull-lengthRNA,buttheyieldissignificantlylowerthanexpected,itispossiblethatcontaminantsintheDNAtemplateareinhibitingtheRNApolymerase,ortheDNAconcentrationmaybeincorrect.Alternatively,additionalpurificationofDNAtemplatemayberequired.Phenol-chloroformextractionisrecommended(seetemplateDNApreparationsection). LowYieldofShortTranscript Highyieldsofshorttranscripts(<0.3kb)areachievedbyextendingincubationtimeandincreasingtheamountoftemplate.Incubationofreactionsupto16hours(overnight)orusingupto2μgoftemplatewillhelptoachievemaximumyield. RNATranscriptSmearingonDenaturingGel IftheRNAappearsdegraded(e.g.smeared)ondenaturingagaroseorpolyacrylamidegel,DNAtemplateiscontaminatedwithRNase.DNAtemplatescontaminatedwithRNasecanaffectthelengthandyieldofRNAsynthesized(asmearbelowtheexpectedtranscriptlength).IftheplasmidDNAtemplateiscontaminatedwithRNase,performphenol/chloroformextraction,thenethanolprecipitateanddissolvetheDNAinnuclease-freewater(seetemplateDNApreparationsection).  RNATranscriptofLargerSizethanExpected IftheRNAtranscriptappearslargerthanexpectedonadenaturinggel,templateplasmidDNAmaybeincompletelydigested.EvensmallamountsofundigestedcircularDNAcanproducelargeamountsoflongtranscripts.Checktemplateforcompletedigestion,ifundigestedplasmidisconfirmed,repeatrestrictionenzymedigestion. LargersizebandsmayalsobeobservedwhentheRNAtranscriptisnotcompletelydenaturedduetothepresenceofstrongsecondarystructures. RNATranscriptofSmallerSizethanExpected Ifdenaturinggelanalysisshowsthepresenceofsmallerbandsthantheexpectedsize,itismostlikelyduetoprematureterminationbythepolymerase.SomesequenceswhichresembleT7RNAPolymeraseterminationsignalswillcauseprematuretermination.Incubatingthetranscriptionreactionatlowertemperatures,forexampleat30°C,mayincreasetheproportionoffull-lengthtranscript,howevertheyieldwillbedecreased.ForGCrichtemplates,ortemplateswithsecondarystructures,incubationat42°Cmayimproveyieldoffull-lengthtranscript.  IfprematureterminationoftranscriptionisfoundinhighspecificactivityradiolabeledRNAprobesynthesis,increasetheconcentrationof“limitingNTP”.Additional“cold”NTPcanbeaddedtothereactiontoincreasetheproportionoffull-lengthtranscript,howevertheimprovementinyieldoffull-lengthproductwillcompromisethespecificactivityofthep TechTips Itisimportanttomixeachcomponentwellbeforesettingupreactions. Makesurereactionsarethoroughlymixed.WerecommendincubatingthereactionsinadryairincubatororinaPCRmachine.  Citations&TechnicalLiterature Citations ProductCitationToolPoweredbyBiozSeemoredetailsonBiozAdditionalCitationsLee,NC.,Larionov,V.,Kouprina,N.(2015)HighlyefficientCRISPR/Cas9-mediatedTARcloningofgenesandchromosomallocifromcomplexgenomesinyeastNucleicAcidsRes;43(8),e55.PubMedID:25690893Jaitin,DA.,Kenigsberg,E.,Keren-Shaul,H.,Elefant,N.,Paul,F.,Zaretsky,I.,Mildner,A.,Cohen,N.,Jung,S.,Tanay,A.andAmit,I.(2014)Massivelyparallelsingle-cellRNA-seqformarker-freedecompositionoftissuesintocelltypes.Science;343,776-779.PubMedID:24531970 Quality,Safety&Legal QualityControlAssays QualityControltestsareperformedoneachnewlotofNEBproducttomeetthespecificationsdesignatedforit.SpecificationsandindividuallotdatafromtheteststhatareperformedforthisparticularproductcanbefoundanddownloadedontheProductSpecificationSheet,CertificateofAnalysis,datacardorproductmanual.FurtherinformationregardingNEBproductqualitycanbefoundhere. Specifications&ChangeNotifications Specifications&ChangeNotificationsTheSpecificationsheetisadocumentthatincludesthestoragetemperature,shelflifeandthespecificationsdesignatedfortheproduct.Thefollowingfilenamingstructureisusedtonamethesedocumentfiles:[ProductNumber]_[Size]_[Version]E2040S_v1 CertificateofAnalysis TheCertificateofAnalysis(COA)isasigneddocumentthatincludesthestoragetemperature,expirationdateandqualitycontrolsforanindividuallot.Thefollowingfilenamingstructureisusedtonamethesedocumentfiles:[ProductNumber]_[Size]_[Version]_[LotNumber]E2040S_v1_0241803E2040S_v1_10012595E2040S_v1_10013491E2040S_v1_10014774E2040S_v1_10018975E2040S_v1_10021740E2040S_v1_10022756E2040S_v1_10027773E2040S_v1_10031646E2040S_v1_10032241E2040S_v1_10035263E2040S_v1_10032035E2040S_v1_10041536E2040S_v1_10044351E2040S_v1_10045383E2040S_v1_10047695E2040S_v1_10050954E2040S_v1_10054479E2040S_v1_10056892E2040S_v1_10059199E2040S_v1_10061569E2040S_v1_10065045E2040S_v1_10068416E2040S_v1_10073088E2040S_v1_10078059E2040S_v1_10078861E2040S_v1_10082352E2040S_v1_10087061E2040S_v1_10090166E2040S_v1_10093284E2040S_v1_10098040E2040S_v1_10099587E2040S_v1_10100208E2040S_v1_10105808E2040S_v1_10109077E2040S_v1_10114318E2040S_v1_10116523E2040S_v1_10120183E2040S_v1_10123751E2040S_v1_10126799E2040S_v1_10130030E2040S_v1_10133564E2040S_v1_10135706E2040S_v1_10138225E2040S_v1_10139668E2040S_v1_10143402E2040S_v1_10146333E2040S_v1_10148493E2040S_v1_10153465E2040S_v1_10154863E2040S_v1_10155138E2040S_v1_10158087E2040S_v1_10161532 SafetyDataSheets ThefollowingisalistofSafetyDataSheet(SDS)thatapplytothisproducttohelpyouuseitsafely.CTP FLucControlTemplateUTP ATPT7RNAPolymeraseMixGTP 10XT7ReactionBuffer LegalandDisclaimers Thisproductiscoveredbyoneormorepatents,trademarksand/orcopyrightsownedorcontrolledbyNewEnglandBiolabs,Inc(NEB).WhileNEBdevelopsandvalidatesitsproductsforvariousapplications,theuseofthisproductmayrequirethebuyertoobtainadditionalthirdpartyintellectualpropertyrightsforcertainapplications.Formoreinformationaboutcommercialrights,pleaseemailusatbusdev@neb.com.Thisproductisintendedforresearchpurposesonly.Thisproductisnotintendedtobeusedfortherapeuticordiagnosticpurposesinhumansoranimals.NewEnglandBiolabs(NEB)iscommittedtopracticingethicalscience–webelieveitisourjobasresearcherstoasktheimportantquestionsthatwhenansweredwillhelppreserveourqualityoflifeandtheworldthatwelivein.However,thisresearchshouldalwaysbedoneinsafeandethicalmanner.Learnmore. 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