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NEB/ProtoScript® First Strand cDNA Synthesis Kit/E6300S/150 reactions
  • NEB/ProtoScript® First Strand cDNA Synthesis Kit/E6300S/150 reactions

NEB/ProtoScript® First Strand cDNA Synthesis Kit/E6300S/150 reactions

价格: ¥7200.00 市场价: 12000.00

品牌: NEB
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    • Description:

      ProtoScript® FirstStrandCDNASynthesisKit featurestwooptimizedmixes,M-MuLVEnzymeMixandM-MuLVReactionMix.M-MuLVEnzymeMixcombinesM-MuLVReverseTranscriptaseandMurineRNaseInhibitor,whileM-MuLVReactionMixcontainsdNTPsandanoptimizedbuffer.Thekitalsocontainstwooptimizedprimersforreversetranscriptionandnuclease-freewater.Ananchoredoligo-dTprimer[d(T)23VN]forcestheprimertoannealtothebeginningofthepolyAtail.TheoptimizedRandomPrimerMixprovidesrandomandconsistentprimingsitescoveringtheentireRNAtemplateincludingbothmRNAsandnon-polyadenylatedRNAs.ThefirststrandcDNAproductgeneratedismorethan10kb(Figure1).

      GeneralInformationforSuccessfulcDNASynthesis:

      TemplateRNA
      IntactRNAofhighpurityisessentialforsensitiveRT-PCRdetection.RNAshouldhaveaminimumA260/A280ratioof1.7orhigher. 

      EithertotalRNAormRNAcanbeusedinthereversetranscriptionreaction.TotalRNAisgenerallysufficientformostRT-PCRanalyses.However,ifdesiredmRNAcanbeeasilyobtainedusingaPolyASpinmRNAIsolationKit(NEB#S1560)orMagneticmRNAIsolationKit(NEB#S1550).

      TheamountofRNArequiredfordetectiondependsontheabundanceofthetranscriptofinterest.Ingeneral1ngto1μgtotalRNAor0.1-100ngmRNAarerecommended.

      FirstStrandcDNASynthesisReaction
      DenaturationofRNAandprimerat70°Cfor5minutescanremovesecondarystructuresthatmayimpedelongcDNAsynthesis.However,thisstepcanbeomittedinsomecases(unpublishedresults). 

      Werecommendincubationat42°Cforonehourformaximumyieldandlength.However,manytargetscanbedetectedafteramuchshorterincubationtime.Forexample,5minutesincubationisenoughfora2kbcDNAsynthesis.

      ChoiceofPrimersforReverseTranscription
      Oligod(T)primingispreferredformostapplicationsbecauseitensuresthatallcDNAcopiesterminateatthe3´endofthemRNAandproducesthelongestcontiguouscDNA.Ananchoredoligo-d(T)primer[d(T)23VN]forcestheprimertoannealtothestartofthepolyAtail,therebypreventingprimingatinternalsitesinthepolyAtail(1).However,twootherprimingchoicesarepossIBLeifdesired.

      TheRandomPrimerMixisanoptimizedmixofhexamerandd(T)23VNprimers.ItprovidesrandomprimingsitescoveringtheentireRNAtemplatesincludingbothmRNAsandnon-polyadenylatedRNAs(suchasribosomalRNAs).TheRandomPrimerMixyieldsshortercDNAsonaverageandcanbeusedforthedetectionofmultipleshortRT-PCRproducts.RandomPrimerMixoffersgoodperformanceinawiderangeofRNAtemplates. 

      Whenagene-specificprimerisusedinacDNAsynthesisreaction,thecDNAproductcanbeusedonlyforamplificationofthattranscript.ThisprimingmethodgivesgoodresultswhentheamountofRNAislimiting(below10ng)andonlyoneparticularcDNAisdesired.

      Recommendedprimerconcentration:
      PRIMER--Finalconc.
      OLIGOd(T)23VN----5μM
      RANDOMPRIMERMIX--6μM
      SPECIFICPRIMER --0.1–1μM





      Figure1:Figure 1

      FirststrandcDNAsynthesiswascarriedoutwith1XM-MuLVEnzymeMixat42°Cusing2μgofhumanspleentotalRNA.Negativecontrolreactionswerecarriedoutwith1XM-MuLVReactionMix.AfractionofthefirststrandcDNAproductwasusedtoamplifysequencesspecificforthreedifferentmessengerRNAsusing1XLongAmp™Taq2XMasterMix(NEB#M0287).Lane1:1.1kbofbeta-actingene.Lane2:noRTcontrolof1.1kbofbeta-actingene.Lane3:4.7kbofXrn-1gene.Lane4:noRTcontrolof4.7kbofXrn-1gene.Lane5:9.8kbofguaninenucleotideexchangefactorp532.Lane6:noRTcontrolof9.8kbofguaninenucleotideexchangefactorp532.MarkerMis2-LogDNALadder(NEB#N3200).

      KitComponents

      Thefollowingreagentsaresuppliedwiththisproduct:

      Storeat(°C)Concentration
      Oligod(T)23VN-2050μM
      Nuclease-freeWater-20
      M-MuLVEnzymeMix-2010X
      M-MuLVReactionMix-202X
      RandomPrimerMix-2060μM
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