ProductDescription

T7RNAPolymerasecatalysesthesynthesisofRNAinthepresenceofdouble-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:

PreparationoffluorescentorrADIoactivelylabeledRNAprobes
PreparationoflargeRNAtranscriptsforanalysisorinvitrotranslation
AmplificationofRNA(inconjunctionwithAMVRTandRibonucleaseH.)
Preparationofanti-senseRNA
Preparationofribozymes
PreparationofmRNAprecursorforsplicing
PreparationofdsRNAforRNAinterferenceorsilencing
PreparationofsubstrateinRNaseprotectionassays
PreparationoftemplateforgenomicDNAsequencing
PreparationofRNAforstudiesofRNAsecondarystructureandRNA-proteininteractions

Reagentssupplied:

5XReactionBufferforT7RNAPolymerase

1XReactionBufferConditions:

40mMTrisHCl,pH7.7
6mMMgCl2
10mMDTT
2mMSpermidine

Tobesupplementedwith:

0.5mMATP,CTP,GTPandUTP
DNAtemplatewithT7promoter
(20nMofalinearizedplasmidDNAor40ug/mLofa3Kbplasmid)
Incubateat37ºC

UnitDefinition:

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.

QualityControlofT7RNAPolymerase

1.)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.),10.27-10.37
Sambrook,J.,Fritsch,E.F.,andManiatis,T.(1989)MolecularCloning:ALaboratoryManual,(2ndEd.),18.82-18.84

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