RACE product confirmation - (Apr/17/2012 )
my lab mate and I have just finished the RACE experiment for our respective transcripts through TA-cloning and sequencing. Both of us got very good sequence reads from our RACE products with decent sized UTRs (5' 80bp and 3' 180bp). However, the problem is that my buddy told me that the RACE product is not reliable and that we should see if we can extend the transcript further. And when he confirmed the transcript through PCR from his cDNA pool, he found the 5' as well as 3' to be twice longer and still more to be confirmed. So I was wondering if this is possible and if so, then whats the point of doing RACE when we could just make primers and PCR them to check for extension?
Any advices would be helpful!
RACE allows you to see where the product terminates as such and it not ideal for generating the UTRs, but is the best method at the moment. PCR relies on two primers to produce a visible product on a gel. You could take the known sequence and make primers then sequence from the template, which would essentially be performing RACE, but without getting a clonable product (which is usually the point of performing the RACE).