5'-RACE cloning - the sequence of RACE product 5'end (Jul/31/2005 )
i just cloned the 5'RACE product into pBluescript vector with SmaI blunt end cloning.
And i got the 5'end sequence result. but i can't read the sequence of 5'universal primer totally.
i know that would be a little different but should only be some nucleotides?
so i don't know the result correct or not?
(i used BDs SMART RACE kit and sequencing with T7)
thanks a lot!
5' UMP has two sequences, one long, and one short. Most of the PCR products are led by the short primer. So the 5' shoud start with the short primer.
I do not know wheather pBluescript vector has T7 primer. If it has, then you should not use T7 primer to sequence, because the UMP primer in the kit also has T7 primer. The product is a mix and could not be read by sequencer.
Try sequencing from the other end of the insert, or from within your clone. The sequencing primer may be too close to the sequence you want, so the result is not completely clear. The answer will be there, you've just got to get a good sequence result. (I'm not familiar with pBluescript or the 5 RACE kit you used, so can't comment on them, but I agree with bioplant: check that there aren't two T7s!)
And congrats on getting the 5' end!
Thanks for the suggestions of bioplant and nicole!
i'll try to sequence from another end or try not to use T7!