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Optimizedreversetranscriptaseandbuffersystemfortheproductionoffull-lengthCDNA.
- Synthesizefull-lengthcDNA(>15kb)
- 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,PCRproductfromcDNAsynthesizedbyEpicentre"sMMLVHPRT.
TargetTranscript(Size) | 3´/5´Ratios | ||
EpicentreMMLVHighPerformanceReverseTranscriptase | CompetitorI(RNaseH-MutantofMMLVRT) | CompetitorP(MMLVRT) | |
ACTB(1,792b) | 0.9 | 1.7 | 1.2 |
GUSB(2,162b) | 1.0 | 6.1 | 2.5 |
TFRC(5,010b) | 5.5 | 12.1 | 11.3 |
Table1.3´/5´ratioanalysisofcDNAproducedbydifferentreversetranscriptaseenzymes. TotalcellularRNAfromHeLacellswasconvertedtocDNAusingthethreereversetranscriptaseenzymesindicatedinthetable.A3´/5´ratioequalto1.0meansthatequalamountsofPCRproductsareobtainedfromboththe3´and5´endofthecDNAandthereforeisagoodindicationthatthereversetranscriptasehasproducedafull-lengthcDNAcopyofthemRNA.
2A.
2B.
EpicentreMMLV1st-StrandcDNASynthesisKit
CompanyIRNaseH-minusMMLVRT
CompanyPMMLVRT
Figure2.cDNAproducedbyEpicentre"sMMLVHighPerformanceReverseTranscriptaseyieldsasignificantlyimproved3´/5´ratiothancompetitivereversetranscriptases. Theapproximately2160-baseHeLaβ-glucuronidasemRNA(GUSB)wasreversetranscribedintocDNAusingEpicentre"sMMLVHighPerfomanceReverseTranscriptaseandtwocompetitivereversetranscriptaseenzymes.PCRprimerpairstothe3´-endand5´-endofGUSBcDNAweresynthesizedandqPCR(SYBR® GreenIdyedetection)wasperformedusingeachprimerpairandtheGUSBcDNAsastemplates.The3´/5´ratiowascalculatedforeachasdescribedinthetext.(A).PCRampliconsfromthe3´endand5´endoftheGUSBcDNA.(B).qPCRquantificationgraphsfordetectingthe3´ampliconand5´ampliconofGUSBcDNAproducedbyEpicentre"sMMLVHighPerformanceReverseTranscriptaseKitandtwoothercommerciallyavailablereversetranscriptaseenzymes.
ORDERINFORMATION
MMLVHighPerformanceReverseTranscriptase,10XReactionBuffer,DTT. 美国Lucigen公司,自1998年成立至今,一直致力于生命科学领域相关科研产品的研究与开发,在分子生物学领域处于领导性地位。Lucigen公司主要开发各类用于基因克隆的试剂盒及相关产品,包括:CloneSmart®平端克隆试剂盒、BigEasy®线性克隆系统、pEZSeq™平端克隆试剂盒、ClonePlex™ AK文库构建试剂盒、DNA Terminator ®末端修复试剂盒、EconoTaq® DNA聚合酶及E.cloni感受态细胞等。Lucigen公司凭借其独到的产品技术,过硬的产品质量,良好的产品服务赢得了全球广大用户的信赖。-
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