Optimizedreversetranscriptaseandbuffersystemfortheproductionoffull-lengthcDNA.

  • Synthesizefull-lengthcDNA(>15kb)Formerly from Epicentre
  • Amplifyfirst-strandcDNAfrompicogramamountsoftotalRNA
  • OptimizetheRTreactiontoyourspecificneedsbyusingthefirststrandcDNAsynthesisprimers,dNTPsandRNaseinhibitorofyourchoice(notincluded).

TheMMLVHPRTdemonstratessignificantlygreaterreversetranscriptaseactivitythanothercommerciallyavailableMMLVRTenzymes.Typically,just100unitsofMMLVHPRTarerequiredforfull-lengthcDNAsynthesiscomparedto200unitsofcompetitiveMMLVRTenzymes.MMLVHPRTincludesa10XReactionBuffer,optimizedforsynthesisoffull-lengthcDNAfromlongRNAtemplates,andDTT.Theenzyme,bufferandDTTarethesamecomponentsusedintheMMLVReverseTranscriptase1st-StrandcDNASynthesisKit.Byprovidingthesethreecomponentsindividually,youhavetheflexibilitytochoosedNTPs,RNaseInhibitors,etc.andoptimizetheRTreactionforyourspecificneeds.

Figure1.MMLVHPRTproducesfull-lengthcDNAfrommRNAlongerthan15kb. TotalRNAisolatedfromHeLacellswasreversetranscribedandthecDNAwasamplifiedbyPCR.Detectionofthe1.3-kbPCRampliconfromnearthe5´endofthemRNAdemonstratesfull-lengthreversetranscriptionofHERC1mRNA(A).AgarosegelanalysisofthePCRproductsshowsthe1.3-kbampliconfromthe5´endofthemRNA(B).LaneM,100bpDNAladder;lane1,no-RTcontrolreaction;lane2,PCRproductfromcDNAsynthesizedbyEpicentresMMLVHPRT.

TargetTranscript(Size)3´/5´Ratios
EpicentreMMLVHighPerformanceReverseTranscriptaseCompetitorI(RNaseH-MutantofMMLVRT)CompetitorP(MMLVRT)
ACTB(1,792b)0.91.71.2
GUSB(2,162b)1.06.12.5
TFRC(5,010b)5.512.111.3

Table1.3´/5´ratioanalysisofcDNAproducedbydifferentreversetranscriptaseenzymes. TotalcellularRNAfromHeLacellswasconvertedtocDNAusingthethreereversetranscriptaseenzymesindicatedinthetable.A3´/5´ratioequalto1.0meansthatequalamountsofPCRproductsareobtainedfromboththe3´and5´endofthecDNAandthereforeisagoodindicationthatthereversetranscriptasehasproducedafull-lengthcDNAcopyofthemRNA.


2A.

2B.

EpicentreMMLV1st-StrandcDNASynthesisKit

CompanyIRNaseH-minusMMLVRT

CompanyPMMLVRT

Figure2.cDNAproducedbyEpicentresMMLVHighPerformanceReverseTranscriptaseyieldsasignificantlyimproved3´/5´ratiothancompetitivereversetranscriptases. Theapproximately2160-baseHeLaβ-glucuronidasemRNA(GUSB)wasreversetranscribedintocDNAusingEpicentresMMLVHighPerfomanceReverseTranscriptaseandtwocompetitivereversetranscriptaseenzymes.PCRprimerpairstothe3´-endand5´-endofGUSBcDNAweresynthesizedandqPCR(SYBR® GreenIdyedetection)wasperformedusingeachprimerpairandtheGUSBcDNAsastemplates.The3´/5´ratiowascalculatedforeachasdescribedinthetext.(A).PCRampliconsfromthe3´endand5´endoftheGUSBcDNA.(B).qPCRquantificationgraphsfordetectingthe3´ampliconand5´ampliconofGUSBcDNAproducedbyEpicentresMMLVHighPerformanceReverseTranscriptaseKitandtwoothercommerciallyavailablereversetranscriptaseenzymes.

ORDERINFORMATION

MMLVHighPerformanceReverseTranscriptase,10XReactionBuffer,DTT.

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发布于 : 2019-08-14 阅读(0)
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