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.ebiomall.com
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多谢……
各位大侠：小弟最近做个长片段的CDNA与T载体的连接，片段大小6.4K，载体选用的是pUC19和pGEMT-easy,反转录酶用的是RevertAidTirstStrandcDNASynthesisKit(#K1621)，CDNA第二链的扩增用的是LATaq。我是用纯病毒的RNA做模板，操作步骤严格按照说明做的，也曾经有一次获得过电泳图清晰显示的是全长6.4KB左右的片段，与载体连接，结果筛选到的几个克隆，插入的片段仅有2KB左右，奈何？可最近几次再用此反转录试剂盒却不能得到所需片段。在此，小弟想问问大家，你们是否遇到同样的问题，是如何解决的？所用的试剂（载体和酶的选择）和方法是什么，可以告诉后来者，共同进步吧。
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