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First-strandCDNASynthesisKit25ReactionKit.
CONTENTS:
Thiskitcontainssufficientreagentstoperform25firststrandcDNAsyntheseswithupto2mgofpurifiedpolyA(+)mRNAor10mgoftotalcellRNAperreaction.
STORAGE:
12monthsat-20°C
12monthsat-70°C
DESCRIPTION:
ThisFirst-strandcDNASynthesiskithasbeendevelopedtomaximizethesynthesisoffull-lengthcDNA.ThiskitproducesanoptimaltemplateforDNAamplificationtechniques.Testedagainsta7.5kbsyntheticpolyA(+)RNAanda9.8kbnativeviralmRNA,32%and42%ofthecDNAssynthesizedwerefull-length,respectively.Also,cDNAstoapolyA(+)RNAladderconsistedofapproximately70%fulllengthproduct(Fig.1).Theuseoffull-lengthcDNAtemplatesforpolymerasechainreaction(PCR)(1)affordsequalavailABIlityofallsequencesforprimingandamplificationregardlessoflocation.ThiskitincludesRNaseinhibitor,DTTsolution,5Xreactionbuffercontainingnucleotides,andAMVreversetranscriptaseenzyme.Pd(T)12-18andrandomhexamerprimersarealsoprovided,togetherwithRNase-freewaterandapolyadenylatedRNAladderascontroltemplate.
cDNASYNTHESIS
Figure1.[32P]-labeledcDNAtothreeRNAtemplates:
Lanes1and2:7.5kbsyntheticpolyA(+)RNAprimedwithpd(T)12-18andrandomhexamers,respectively.
Lanes3and4:9.8kbtobaccoveinmottlingvirus(TVMV)mRNA,primedwithpd(T)12-18andrandomhexamers,respectively.
Lanes5and6:polyA(+)RNAladder*primedwithpd(T)12-18andrandomhexamers,respectively.
*ThispolyA(+)RNAladderisdifferentfromtheonesuppliedinthiskit.
FORLABORATORYUSEONLY
Downloaddetailsandprotocol.
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