Posted 13 February 2004 - 03:13 PM
Is anyone very familiar with 5'RACE? I am using Invitrogen kit, but it never worked. Looks like RT worked fine since PCR on the RT product using gene specific primers that were designed from the known sequence amplified an expected fragment. But, after nested PCR, I can't get any product (even no smear at all) although I tried to change PCR conditions. Does anyone have any idea about this problem?
Posted 16 February 2004 - 01:16 PM
Dose the positive control coming with the kit work? Did you use gene-specfic primer for the synthesis of first strand cdna? Also make sure the known farthest 5' sequence of your gene is correct (sometimes it is not, if it is of genomic sequence, primer designed on it may not work.)
Once I got my 5' race working by trying kit from several vendors. Clontech sells 5' race ready cDNA which has adaptor linked.
Posted 16 February 2004 - 04:11 PM
Thank you for your reply.
The positive control worked fine. I used gene-specific primer for RT reaction.
As the protocol suggests, I tried increasing temperature for RT (up to 50 degrees), but it didn't help. Is A tailing better than C tailing? (I found that another company uses A) Also, will it be more problematic if the gene is expressed at a very low level?
Posted 16 February 2004 - 10:00 PM
If your gene is expressed at low level, this could be a problematic for RACE.