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Method 5'-RACE

5'-RACE
Method 5'-RACE
Description In the case of promoter trap, 5’-RACE (Rapid Amplification of cDNA Ends) is generally used for the identification of the trapped gene. RNA is extracted from the embryonic stem cell, the reverse transcriptase reacts with anti sense primer (SP1) in the reporter gene of the trap vector, and 1st strand cDNA is synthesized. After polyA tail addition to the 3’end of 1st strand cDNA, 1st PCR is done with oligo dT-anchor primer and anti sense primer (SP2). 2nd PCR is continuously done with anchor primer and SP3 primer. The sequence of 2nd PCR product (RACE product) is determined by SP4 primer. It should be a fusion transcript of trapped gene and reporter gene.