Description | ExCellscriptThermostableM-MuLVReverseTranscriptaseisaDNApolymerasewhichutilizesRNAasasubstrateandexhibitsnomeasurableproofreADIng3′→5′exonucleasefunction.ThisthermostablereversetranscriptasecanperformCDNAsynthesisreactionsthroughoutawiderangeoftemperaturesfrom42°Cto72°C. |
Applications | - FirststrandcDNAsynthesisforRT-PCRandreal-timeRT-PCR
- SynthesisofcDNAforcloningandexpression
- GenerationoflabeledcDNAprobesformicroarrays
- DNAlabeling
- AnalysisofRNAbyprimerextension
- IsothermalnucleicacidamplificationsuchasLAMP
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Source | ArecombinantE.colistraincarryinganengineeredMoloney-MurineLeukemiaVirusReverseTranscriptasegene |
QualityControl | - Theabsenceofendo-,exodeoxyribonucleases,phosphatases,andribonucleasesconfirmedbyappropriatequalitytests.
- FunctionallytestedinfirststrandcDNAsynthesis.
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Unitdefinition | 1unitisdefinedastheamountofenzymerequiredtoincorporate1nmolofdTTPintoacidinsolublematerialin10minutesat37°Cusingpolyr(A)/oligo(dT)asasubstrate. |
Components | - 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
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Protocol | - PrimerAnnealing:CombinethefollowinginanRNase-freereactionvessel:
Amount | Description | FinalConcentration | 1µL | 25mMdNTPSolution(N2050L | 2.0mM | XµL | 1ng-2µgTotalRNA-or- | | XµL | 5-500ngmRNA(polyAselected) | | 1µL | Oligo(dT)12-18(500µg/ml)-or- | 40µg/mL | 1µL | RandomPrimers(125µg/ml)-or | 10µg/mL | 1µL | GSPPrimer(2pmol) | 165µM | XµL | SterileWater | N/A | 10µL | TotalVolume |
- Heatreactionfor5minutesat65°C.Spinbriefly(5sec)topulldowncondensateandplaceimmediatelyonice.
- Add1µL10XM-MuLVRTBufferandDNase-freewatertoafinalvolumeof10µLperreaction.
- Incubate:
- IfusingOligo(dT)orGSPprimers:2minutes@42°C
- IfusingRandomprimers:2minutes@25°C
- Add1µL(200units)thermostableM-MuLVReverseTranscriptaseandmixbygentlypipettingsample.(Note:ifusingrandomprimers,pre-incubatereaction@25°Cfor10minutes).
- Incubateat42~72°Cfor45-60minutes.
- Inactivateenzymeat95°Cfor10minutes.
- Storeproductsat-20°Corproceedtonextstep.
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Shipping | 4°C |
Note | Forresearchuseonly |