Description:
QuantumScript™HDReverseTranscriptaseisanengineeredversionwithincreasedsensitivity,improvedspecificityandmaximumthermostability.QuantumScript™HDReverseTranscriptasehasbeenengineeredtohavelongerhalflifeat50°C,whichenablesitsabilitytoprocesslongerRNAwithmorecomplexedsecondarystructures. Enhancedthermostabilityofthisenzymeisobtainedthroughre-engineeredRNA-basedDNAPolymerasedomainandthefusionofanovelRNA-interactingsurfacedomainattheRNaseHdomainsite.Theenzymeispurifiedtohomogeneitytoensurehighthermostability,specificity,fidelity,yield,andmorefulllengthcDNAsynthesisthatthepremiumreversetranscriptaseprovides.Theoptimalfist-strandcDNAsynthesistemperatureforthisenzymeis50°C,andithasabroadworkingtemperaturerangefrom37°Cto55°C,withcDNAproductsizefrom100bpto12Kb. CatalogNo.
SSIII-100,SSIII-200andSSIII-300
Source
E.coli
Concentration
200u/μl
StorageBuffer
20mMTris-HCl(pH7.5),1mMDTT,0.05%(v/v)TritonX-100,0.1mMEDTA,0.1MNaCland 50%(v/v)glycerol.
ReactionBuffer(5x)
250mMTris-HCl(pH8.3),375mMKCl,15mMMgCl2,and50mMDTT
UnitDefinition
Oneunitoftheenzymeincorporates1nmoleofdTTPintoacid-precipitablematerialin10minutesat37˚Cusingpoly(A):oligo(dT)25astemplate-primer.
QualityControl
Thisenzymehaspassedthequalitycontrolassays:SDS-PAGEanalysisforpurity,functionalabsenceofendonucleaseactivities,functionalabsenceofexonucleaseactivities,functionalabsenceofproteaseactivity. StorageandHandling:-20°C Protocol
First-StrandcDNASynthesis
MaterialstoBeSuppliedbytheUser - RNAseInhibitor(Cat.#RNIN-100,RNIN-200,RNIN-300)
- dNTP,10mM(Cat.#dNTP-10M,dNTP-25M)
- Nuclease-FreeWater
Thefollowingprocedureuses10pgto5µgoftotalRNAor10pgto500ngofmRNA. - InasterileRNase-freemicrocentrifugetube,addprimers (200-500ngofoligo(dT)12-18,50-250ngofrandomprimersor2pmolofspecificprimers).Heatthetubeto70°Cfor5minutesandincubateonicefor1mintodenatureanypossIBLesecondarystructures withinthetemplate.Spinbrieflytocollectthesolutionatthebottomofthetube.
- Addthefollowingcomponentstotheannealedprimer/templateintheordershown.
Note:DonotaltertheratioofprimertomRNA.
5µl5XReactionBuffer;5µlof10mMdNTPmixture(10mMeachdATP,dGTP,dCTPanddTTP)
25unitsRNAseinhibitor
0.5µlQuantumScriptReverseTranscriptase(200u/μl)
Addnuclease-freewatertothefinalvolumeof25µl - Mixgently.Forrandomprimers,incubatetubeat25°Cfor5min.Performfirst-strandsynthesisat55°Cfor30-60min.Reactiontemperaturemaybeoptimizedbetween50°C-60°Cfordifficulttemplatewithhighsecondarystructure.
- Inactivatetheenzymebyincubationat70°Cfor15min.
- WhenperformPCRamplificationafterstep4,removalofRNAishighlyrecommendedpriortothePCRamplificationtoensuretheyieldofPCRproduct.Additionof2unitsofRNaseH(Cat.#RNHE-100,RNHE-200,RNHE-300)and20minincubationat37°CisrecommendedfortheremovalofRNA.Standardprotocolsforsecond-strandsynthesismaybefoundinreference2.
Note:The5XReactionBufferiscompatiblewithenzymesusedinanumberofdownstreamapplications.TypicallythereisnoneedforphenolextractionsorethanolprecipitationsusingthisprotocolbeforeanyPCRamplification. References - Roth,M.J.,Tanese,N.andGoff,S.P.(1985)PurificationandcharacterizationofmurineretroviralreversetranscriptaseexpressedinEscherichiacoli.J.Biol.Chem.260,9326–35.
- Sambrook,J.,Fritsch,E.F.andManiatis,T.(1989)In:MolecularCloning:A;LaboratoryManual,ColdSpringHarborLaboratory,ColdSpringHarbor,NewYork,8.64.
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