新型逆转录酶性能参数与质量控制分析.pdfVIP

QuantumScriptTMReverseTranscriptaseisanewengineeredversionofM-MLVreverse

transcriptasewithminimumRNaseHactivityandenhacedthermalstability.Theenzymeis

purifiedtonearhomogeneitytoensurehighperformance.Theoptimalfist-strandcsynthesis

temperatureforthisenzymeis42oC,withcproductsizefrom100bpto7Kb.

CatalogNo.SSII-100,SSII-200andSSII-300

SourceE.coli

Concentration200u/μl

StorageBuffer20mMTris-HCl(pH7.5),1mMDTT,0.05%(v/v)TritonX-100,0.1mM

EDTA,0.1MNaCland50%(v/v)glycerol.

ReactionBuffer(5x)250mMTris-HCl(pH8.3),375mMKCl,15mMMgCl,and50mMDTT

2

UnitDefinitionOneunitoftheenzymeincorporatesInmoleofdTTPintoacid-precipitable

materialin10minutesat37˚Cusingpoly(A):oligo(dT)25astemte-primer.

QualityControlThisenzymehaspassedthequalitycontrolassays:SDSanalysisforpurity,

functionalabsenceofendonuclease/nickaseactivities,functionalabsenceof

exonucleaseactivities,functionalabsenceofproteaseactivity.

StorageandHandling-20˚C

Protocol

First-StrandcSynthesis

MaterialstoBedbytheUser

•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-

250ngofrandomprimeror2pmolofspecificprimers).Heatthetubeto70°Cfor5minutes

andincubateonicefor1mintodenatureanypossiblese