Excellgen/ExcellScript Thermostable Reverse Transcriptase/EG1029/40,000 Units

ExcellScriptThermostableM-MuLVReverseTranscriptaseisaDNApolymerasewhichutilizesRNAasasubstrateandexhibitsnomeasurableproofreading3′→5′exonucleasefunction.ThisthermostablereversetranscriptasecanperformcDNAsynthesisreactionsthroughoutawiderangeoftemperaturesfrom42°Cto72°C.

• FirststrandcDNAsynthesisforRT-PCRandreal-timeRT-PCR
• SynthesisofcDNAforcloningandexpression
• GenerationoflabeledcDNAprobesformicroarrays
• DNAlabeling
• AnalysisofRNAbyprimerextension
• IsothermalnucleicacidamplificationsuchasLAMP

• Theabsenceofendo-,exodeoxyribonucleases,phosphatases,andribonucleasesconfirmedbyappropriatequalitytests.
• FunctionallytestedinfirststrandcDNAsynthesis.

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

• ThermostableM-MuLVReverseTranscriptase:200units/µLin50mMTris-HCl,150mMNaCl,0.1mMEDTA,1mMDTT,0.1%NP-40,50%glycerol,pH7.6@25°C
• 10XReactionBuffer:500mMTris-HCl,750mMKCl,30mMMgCl₂,100mMDTTpH8.3@25°C

1. PrimerAnnealing:CombinethefollowinginanRNase-freereactionvessel:

1. Heatreactionfor5minutesat65°C.Spinbriefly(5sec)topulldowncondensateandplaceimmediatelyonice.
2. Add1µL10XM-MuLVRTBufferandDNase-freewatertoafinalvolumeof10µLperreaction.
3. Incubate:
   • IfusingOligo(dT)orGSPprimers:2minutes@42°C
   • IfusingRandomprimers:2minutes@25°C
4. Add1µL(200units)thermostableM-MuLVReverseTranscriptaseandmixbygentlypipettingsample.(Note:ifusingrandomprimers,pre-incubatereaction@25°Cfor10minutes).
5. Incubateat42~72°Cfor45-60minutes.
6. Inactivateenzymeat95°Cfor10minutes.
7. Storeproductsat-20°Corproceedtonextstep.