首页 > Lifesci > Lifesci/T7 RNA Polymerase in Glycerol/T7G-70000/70000 u

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Lifesci/T7 RNA Polymerase in Glycerol/T7G-70000/70000 u

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T7G-70000

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ProductDescriptionT7RNAPolymerasecatalysesthesynthesisofRNAinthepresenceofdouble-strandedDNAcontainingaT7promotersequence.ItisisolatedfromanE.colitransformedbyaplasmidcontainingtheT7RNAPolymerasegene.Thisenzymeisprovidedinglycerol-basedstoragebuffercontaining:20mMpotassiumphosphatepH7.5,100mMNaCl,1.0mMEDTA,10mMDTT,0.2%TritonX-100and50%glycerol.Alternatively,itisalsoprovidedinthesamebuffercontaining1.0Mtrehaloseinsteadofglycerol(suitableforlyophilization).Concentration70,000units/mL.Pleaseinquireforcustomconcentrationsandbulkquantities.Applications:PreparationoffluorescentorrADIoactivelylabeledRNAprobesPreparationoflargeRNAtranscriptsforanalysisorinvitrotranslationAmplificationofRNA(inconjuctionwithAMVRTandRibonucleaseH.)Preparationofanti-senseRNAPreparationofribozymesPreparationofmRNAprecursorforsplicingPreparationofdsRNAforRNAinterferenceorsilencingPreparationofsubstrateinRNaseprotectionassaysPreparationoftemplateforgenomicDNAsequencingPreparationofRNAforstudiesofRNAsecondarystructureandRNA-proteininteractionsReagentssupplied:5XReactionBufferforT7RNAPolymerase1XReactionBufferConditions:40mMTrisHCl,pH7.76mMMgCl210mMDTT2mMSpermidineTobesupplementedwith:0.5mMATP,CTP,GTPandUTPDNAtemplatewithT7promoter(20nMofalinearizedplasmidDNAor40ug/mLofa3Kbplasmid)Incubateat37ºCUnitDefinition:Oneunitisdefinedastheamountofenzymethatwillincorporate3nmolofUridinetriphosphateintoacid-insolubleforminonehourat37ºCunderstandardassayconditions:40mMTrisHCl,pH7.5,30mMMgCl2,20mMDTT,0.4mMGTP,CTP,ATP,UTPand[3H]-UTP,20µg/mLnon-linearizedplasmidDNA,50µg/mLBSA.GeneralNotes:DithiothreitolisrequiredforT7RNAPolymeraseactivity.ThislABIlecomponentofthereactionbuffermayberestoredbysupplementingreactionswithafinalconcentrationof10mMDTT.HigheryieldsofRNAmaybeobtainedbyincreasingtheconcentrationofNTP’stoasmuchas4mM.Caremustbetakenthatthetotalsaltconcentrationinthereactiondoesnotexceed50mM,sincethisenzymeissensitivetosaltconcentrationsexceedingthisamount.QualityControlofT7RNAPolymerase1.)DNaseActivity:One-halfµgofHaefragmentsofPhiX-174DNAisincubatedat37ºCwith175unitsofT7RNAPolymerasefor3hours,andthenelectrophoresedinanativeagarosegelsimultaneouslywithcontrolpositiveDNase1reactions.Nomorethantheequivalentof1.25X10E-4unitofDNase1isdetected.2.)RibonucleaseActivity:OnemicrogramofanRNALadderisincubatedfor2hourswith280unitsofT7RNAPolymerase,andthenelectrophoresedinanativeagarosegelsimultaneouslywithcontrolpositiveRNase1Areactions.Nomorethantheequivalentof8.0X10E-8unitofRNase1Aisdetected.3.)SpecificActivity:ThespecificactivityoftheT7RNAPolymeraseisnolessthan400,000unitspermg.References:Sambrook,J.,Fritsch,E.F.,andManiatis,T.(1989)MolecularCloning:ALaboratoryManual,(2ndEd.),10.27-10.37Sambrook,J.,Fritsch,E.F.,andManiatis,T.(1989)MolecularCloning:ALaboratoryManual,(2ndEd.),18.82-18.84