1、5RACE试剂盒原理及注意事项,SMARTer RACE 5/3 Kit From TaKaRa Code:624858,原理,SMARTScribe Reverse Transcriptase SMARTer II A Oligonucleotide （-70）,5RACE First-Strand cDNA Synthesis 试剂清单,10 l SMARTer II A Oligonucleotide (24 M)(-70) 10 l 5 RACE CDS Primer A (12 M) 40 l 5X First-Strand Buffer (RNAse-Free) 5 l Dithi
2、othreitol (DTT) (100 mM) 1 ml Deionized H2O 10 l RNase Inhibitor (40 U/l) 20 l SMARTScribe Reverse Transcriptase (100 U/l) 10 l dNTP mix (20 mM),5RACE PCR 试剂清单,400 l 10X Universal Primer A Mix (UPM)（混合引物） 50 l Universal Primer Short (10 M)(已合成) 1.25 ml Seq Amp PCR Buffer (2X)（混合试剂） 50 l SeqAmp DNA P
3、olymerase(已用完),GSP引物设计,23-28bp, 50%-70%GC Tm 65C, best results Tm 70C Product range: 1-3kb Add GATTACGCCAAGCTT to 5 ends（linearized pRACE vector） Nested primer Long primer = 5CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAG AGT3 Short primer = 5CTAATACGACTCACTATAGGGC3,RACE-Ready cDNA获得,在全部过程中都使用Tricine-ED
4、TA Buffer稀释cDNA样本 Protocol有变化,20ul,5Rapid Amplification of cDNA Ends (5RACE),Protocol改动,含dNTP,已用完,混合引物,5Rapid Amplification of cDNA Ends (5RACE),Program变化,详细参照试剂盒配套说明书,实验室常用DNA polymerase,Easy Taq AP111 TaKaRa Taq R001B,Protocol,Easy Taq AP111,TaKaRa Taq R001B,Program,Easy Taq AP111,TaKaRa Taq R001B,Additional,两者均在扩增产物3端带A碱基 Easy Taq AP111产物不适用于聚丙烯酰胺凝胶电泳 TAKARA Taq推荐进行cool start,详细参照酶配套说明书,
