5’RACE试剂盒原理及注意事项

5’RACE试剂盒原理及注意事项SMARTer®RACE5’/3’KitFromTaKaRaCode:624858原理•SMARTScribeReverseTranscriptase•SMARTerIIAOligonucleotide（-70℃）5’RACEFirst-StrandcDNASynthesis试剂清单•10μlSMARTerIIAOligonucleotide(24μM)(-70℃)•10μl5'RACECDSPrimerA(12μM)•40μl5XFirst-StrandBuffer(RNAse-Free)•5μlDithiothreitol(DTT)(100mM)•1mlDeionizedH2O•10μlRNaseInhibitor(40U/μl)•20μlSMARTScribeReverseTranscriptase(100U/μl)•10μldNTPmix(20mM)5’RACEPCR试剂清单•400μl10XUniversalPrimerAMix(UPM)（混合引物）•50μlUniversalPrimerShort(10μM)(已合成)•1.25mlSeqAmpPCRBuffer(2X)（混合试剂）•50μlSeqAmpDNAPolymerase(已用完)GSP引物设计•23-28bp,50%-70%GC•Tm≥65°C,bestresultsTm70°C•Productrange:1-3kb•AddGATTACGCCAAGCTTto5’ends（linearizedpRACEvector）•Nestedprimer•Longprimer=5’CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT–3'•Shortprimer=5’–CTAATACGACTCACTATAGGGC–3’RACE-ReadycDNA获得•在全部过程中都使用Tricine-EDTABuffer稀释cDNA样本•Protocol有变化20ul5’RapidAmplificationofcDNAEnds(5’RACE)•Protocol改动含dNTP已用完混合引物5’RapidAmplificationofcDNAEnds(5’RACE)•Program变化•详细参照试剂盒配套说明书实验室常用DNApolymeraseEasyTaqAP111TaKaRaTaqR001BProtocolEasyTaqAP111TaKaRaTaqR001BProgramEasyTaqAP111TaKaRaTaqR001BAdditional•两者均在扩增产物3’端带A碱基•EasyTaqAP111产物不适用于聚丙烯酰胺凝胶电泳•TAKARATaq推荐进行coolstart•详细参照酶配套说明书