SMARTer_RACE_5_3__Kit_User_Manual_022714(4)

clontech试剂盒 SMARTer_RACE 5'/3' Kit User Manual

I. Introduction

The SMARTer RACE 5’/3’ Kit provides a method for performing both 5’- and 3’-rapid amplification of cDNA ends (RACE). The SMARTer RACE 5’/3’ Kit includes our SMARTer II A Oligonucleotide and SMARTScribe Reverse Transcriptase, which provides better sensitivity, less background and higher specificity than previous

kits. This powerful system allows you to amplify the complete 5’ sequence of your target transcript from as little as 10 ng of total RNA. The cornerstone of SMARTer RACE cDNA synthesis is SMART technology, which eliminates the need for problematic adaptor ligation and lets you use first-strand cDNA directly in RACE PCR, a benefit that makes RACE far less complex and much faster (Chenchik et al., 1998). Additionally, the SMARTer RACE Kit exploits Clontech’s technology for suppression PCR & step-out PCR to increase the sensitivity and reduce the background of the RACE reactions. You can use either poly A+ or total RNA as starting material for constructing full-length cDNAs, even of very rare transcripts.

The SMARTer RACE 5’/3’ Kit is an improved version of our original SMARTer RACE cDNA Amplification Kit, designed to accommodate larger RNA input volumes and perform more efficiently on challenging targets

(e.g., those that are long, GC-rich, etc.). RACE PCR products are amplified with our highly robust SeqAmp DNA Polymerase, and cloned into the linearized pRACE vector with In-Fusion® HD Cloning. The In-Fusion HD Cloning Kit, NucleoSpin Gel and PCR Clean-Up Kit, and Stellar Competent Cells are included for your

convenience in cloning RACE products.

SMART technology provides a mechanism for generating full-length cDNAs in reverse transcription reactions (Zhu et al., 2001). This is made possible by the joint action of the SMARTer II A Oligonucleotide and

SMARTScribe Reverse Transcriptase. When the SMARTScribe RT reaches the 5’ end of the RNA, its terminal transferase activity adds a few additional nucleotides to the 3’ end of the first-strand cDNA (Figure 1).

Figure 1. Mechanism of SMARTer cDNA synthesis. First-strand cDNA synthesis is primed using a modified oligo (dT) primer. After SMARTScribe Reverse Transcriptase (RT) reaches the end of the mRNA template, it adds several nontemplated residues. The SMARTer II

A Oligonucleotide anneals to the tail of the cDNA and serves as an extended template for SMARTScribe RT.

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