MCLAB/QuantumScript™ HD Reverse Transcriptase /SSIII-OEM/1 Ea

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.

1. InasterileRNase-freemicrocentrifugetube,addprimers (200-500ngofoligo(dT)_12-18,50-250ngofrandomprimersor2pmolofspecificprimers).Heatthetubeto70°Cfor5minutesandincubateonicefor1mintodenatureanypossIBLesecondarystructures withinthetemplate.Spinbrieflytocollectthesolutionatthebottomofthetube.
2. Addthefollowingcomponentstotheannealedprimer/templateintheordershown.
   Note:DonotaltertheratioofprimertomRNA.
   5µl5XReactionBuffer;5µlof10mMdNTPmixture(10mMeachdATP,dGTP,dCTPanddTTP)
   25unitsRNAseinhibitor
   0.5µlQuantumScriptReverseTranscriptase(200u/μl)
   Addnuclease-freewatertothefinalvolumeof25µl
3. Mixgently.Forrandomprimers,incubatetubeat25°Cfor5min.Performfirst-strandsynthesisat55°Cfor30-60min.Reactiontemperaturemaybeoptimizedbetween50°C-60°Cfordifficulttemplatewithhighsecondarystructure.
4. Inactivatetheenzymebyincubationat70°Cfor15min.
5. WhenperformPCRamplificationafterstep4,removalofRNAishighlyrecommendedpriortothePCRamplificationtoensuretheyieldofPCRproduct.Additionof2unitsofRNaseH(Cat.#RNHE-100,RNHE-200,RNHE-300)and20minincubationat37°CisrecommendedfortheremovalofRNA.Standardprotocolsforsecond-strandsynthesismaybefoundinreference2.

Note:The5XReactionBufferiscompatiblewithenzymesusedinanumberofdownstreamapplications.TypicallythereisnoneedforphenolextractionsorethanolprecipitationsusingthisprotocolbeforeanyPCRamplification.

References

1. Roth,M.J.,Tanese,N.andGoff,S.P.(1985)PurificationandcharacterizationofmurineretroviralreversetranscriptaseexpressedinEscherichiacoli.J.Biol.Chem.260,9326–35.
2. Sambrook,J.,Fritsch,E.F.andManiatis,T.(1989)In:MolecularCloning:A;LaboratoryManual,ColdSpringHarborLaboratory,ColdSpringHarbor,NewYork,8.64.