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Excellgen/ExcellScript MMLV (MMLV) Reverse Transcriptase RNase H minus/EG1061/100,000 Units188bio精品生物—专注于实验室精品爆款的电商平台 - 蚂蚁淘旗下精选188款生物医学科研用品
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Excellgen/ExcellScript MMLV (MMLV) Reverse Transcriptase RNase H minus/EG1061/100,000 Units
Description

ExcellScriptM-MuLVReverseTranscriptaseRNaseHminusisanRNA-dependentDNApolymerasewithnodetectableRNaseHactivity.ExcellScriptcanbeusedtogeneratefirst-strandcDNAfrompolyAmRNAortotalRNAforuseindownstreamapplicationssuchasRT-PCR,cDNAcloningorlibraryconstructionforRNA-Seq.PointmutationsintheRNaseHdomainincreasethethermostabilityoftheenzymeandsupportgreatercDNAyieldoffull-lengthtranscriptsthanwildtypeM-MLVReverseTranscriptase

Applications
  • FirststrandcDNAsynthesisforRT-PCRandreal-timeRT-PCR
  • SynthesisofcDNAforcloningandexpression
  • GenerationoflabeledcDNAprobesformicroarrays
  • DNAlabeling
  • AnalysisofRNAbyprimerextension
SourceArecombinantE.colistraincarryinganengineeredMoloney-MurineLeukemiaVirusReverseTranscriptasegene
UnitDefinition1unitisdefinedastheamountofenzymerequiredtoincorporate1nmolofdTTPintoacidinsolublematerialin10minutesat37°Cusingpolyr(A)/oligo(dT)asasubstrate.
Components
  • M-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
StorageCondition-20°C
Protocol
  1. PrimerAnnealing:CombinethefollowinginanRNase-freereactionvessel:
AmountDescriptionFinalConcentration
1µL25mMdNTPSolution(N2050L2.0mM
XµL1ng-2µgTotalRNA-or-
XµL5-500ngmRNA(polyAselected)
1µLOligo(dT)12-18(500µg/ml)-or-40µg/mL
1µLRandomPrimers(125µg/ml)-or10µg/mL
1µLGSPPrimer(2pmol)165µM
XµLSterileWaterN/A
10µLTotalVolume
  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)M-MuLVReverseTranscriptaseandmixbygentlypipettingsample.(Note:ifusingrandomprimers,pre-incubatereaction@25°Cfor10minutes).
  5. Incubateat42°Cfor45-60minutes.
  6. Inactivateenzymeat85°Cfor10minutes.
  7. Storeproductsat-20°Corproceedtonextstep.

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