EnzScript™ (MMLV Reverse Transcriptase RNase H) | Enzymatics

ProductDescription

EnzScript™(M-MLVReverseTranscriptaseRNaseHminus)isanRNA-dependentDNApolymerasewithnodetectableRNaseHactivity.EnzScript™canbeusedtogeneratefirst-strandCDNAfrompolyAmRNAortotalRNAforuseindownstreamapplicationssuchasRT-PCR,cDNAcloningorlibraryconstructionforRNA-Seq.PointmutationsintheRNaseHdomainincreasetheThermostABIlityoftheenzymeandsupportgreatercDNAyieldoffull-lengthtranscriptsthanwildtypeM-MLVReverseTranscriptase(1).

SourceofProtein:ArecombinantE.colistraincarryingtheMoloney-MurineLeukemiaVirusReverseTranscriptasegenewith3pointmutationsintheRNaseHdomainthateliminatedetectableRNaseHactivity.

UnitDefinition:1unitisdefinedastheamountofenzymerequiredtoincorporate1nmolofdTTPintoacidinsolublematerialin10minutesat37°Cusingpolyr(A)/oligo(dT)asasubstrate.

Molecularweight:75,938Daltons

QualityControlAnalysis

UnitActivity ismeasuredusinga2-foldserialdilutionmethod.Dilutionsofenzymeweremadein1XM-MuLVRTRNAseH-Bufferandaddedto50µLreactionscontaining20µg/mLpolyr(A)RNA,oligo(dT)DNA,1XRTBuffer,3H-dTTPand250µMdTTP.Reactionswereincubated10minutesat37°C,plungedonice,andanalyzedusingthemethodofSambrookandRussell(MolecularCloning,v3,2001,pp.A8.25-A8.26).

ReverseTranscriptasefunction wasdeterminedbytheenzymesabilitytogeneratea9.4kBcDNAtranscript.Following2-StepRT-PCRthe9.4Kbampliconwasvisualizedbyagarosegelelectrophoresis

ProteinConcentration(OD280) isdeterminedbyOD280 absorbance.PhysicalPurityisevaluatedbySDS-PAGEofconcentratedanddilutedenzymesolutionsfollowedbysilverstaindetection.Purityisassessedbycomparingtheaggregatemassofcontaminantbandsintheconcentratedsampletothemassoftheproteinofinterestbandinthedilutedsample.

Single-StrandedExonuclease isdeterminedina50µLreactioncontainingarADIolabeledsingle-strandedDNAsubstrateand10µLofenzymesolutionincubatedfor4hoursat37°C.

Double-StrandedExonuclease isdeterminedina50µlreactioncontainingaradiolabeleddouble-strandedDNAsubstrateand10µLofenzymesolutionincubatedfor4hoursat37°C.

Double-StrandedEndonuclease isdeterminedina50µLreactioncontaining0.5µgofplasmidDNAand10µLofenzymesolutionincubatedfor4hoursat37°C.

E.coli 16SrDNAContamination isevaluatedusing5µLreplicatesamplesofenzymesolutiondenaturedandscreenedinaTaqManqPCRassayforthepresenceofcontaminating E.coli genomicDNAusingoligonucleotideprimerscorrespondingtothe16SrRNAlocus.

Non-SpecificRNAse contaminationisassessedusingtheRNAseAlertkit,(IntegratedDNATechnologies),followingthemanufacturersguidelines.

Suppliedin: 20mMTris-HCl,100mMNaCl,0.1mMEDTA,1mMDTT,0.01%NP-40Alternative,50%glycerol,pH7.5@25°C.

Suppliedwith: 5XM-MLVReverseTranscriptaseRNAseH-ReactionBuffer(B7601)and100mMDTT(B9060)

KitContents
CommonApplications

EnzScript™(M-MLVReverseTranscriptaseRNaseHminus)isanRNA-dependentDNApolymerasecommonlyusedtosynthesizeFirst-StrandcDNAforRT-PCRamplification,cDNAcloningorRNASeq.ReducedRNaseHactivityenablesgreateryieldoffull-lengthcDNAtranscripts(5kb)andincreasedthermalstabilityoverstandardM-MLVRT.

FirstStrandReactionProtocol

GeneralprecautionagainstRNasedegradationoftemplateRNAshouldbetakenwhensettingupFirst-Strandreactionssuchasuseofnuclease-freewater,RNaseinhibitor,RNase-freetubesandsterilepipettipswithfilters.Thefollowingprocedurecanbeusedasaguidelineforpreparinga20µlFirst-StrandcDNAreaction.

MaterialstobeprovidedbyUser:

•Sterile,nuclease-freewater
•Primer(oligodT(15-20) or randomhexamers or gene-specific)
•dNTPmix(dATP,dCTP,dGTP,dTTP)
•RNAtemplate
•RNaseInhibitor

1.AddthefollowingcomponentstoanRNase-freemicrocentrifugetubeonice.Formorethanonereaction,prepareamastermix.

*1ngto1µgtotalRNA or 1to250ngmRNA

2.Heatmicrocentrifugetubeto65°Cfor5minutesandquicklycoolonicefor2minutestoannealprimertoRNAtemplate.Spintubebrieflytocollectcondensate.

3.Addthefollowingcomponents(toeachFirst-Strandreaction)tothemicrocentrifugetubeonice:

25°Cfor2minutes(oligodT(15-20),gene-specificprimer) or 25°Cfor10minutes(randomhexamer)42°Cfor30to60minutesHeatkillat70°Cfor15minutes

5.UsecDNAindownstreamapplicationorstoreat-20°C.ForRT-PCR,1to2µlofcDNAfromFirst-StrandreactionistypicallyaddedastemplatetoPCR.Optional:RemoveRNAstrandpriortoPCRbyadding1µlRNaseH(5U)tocDNA:RNAhybrid,incubateat37°Cfor20minutesand65°Cfor10minutes(heatkill).RNaseHtreatmentisrecommendedforamplificationoflongamplicons(5kB).

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FrequentlyAskedQuestionsandTroubleshooting

ForFrequentlyAskedQuestions(FAQ)andtroubleshootingpleaseclickhere.

PartNumberP7600LPrice$131/packConcentration200,000U/mLUnitSize10,000ULotNumberShipmentdependentAvailableonrequest
SDSPurityn/a>99%SpecificActivityn/a≥280,000U/mgSSExonuclease2,000DSExonuclease2,000DSEndonuclease2,000NoConversionE.coliDNAContamination2,000RNAseContamination2,000NoDetectablenon-specificRNaseFunctionalRT-PCRAssayn/aSynthesisof9.4kbcDNAtranscript

*Foradetailedsummaryofassayconditionsanddata,refertotheQualityControlsAnalysissection.

LimitationsofUse
Thisproductwasdeveloped,manufactured,andsoldforinvitrouseonly.TheproductisnotsuitableforadmiNISTrationtohumansoranimals.SDSsheetsrelevanttothisproductareavailableuponrequest.