RACE - specific primers in first strand synthesis (Sep/03/2004 )
I am making 5' RACE, but have som difficilties with som low transcribed genes. Will it help to make cDNA with gene specific primer. How do I combine the GSP with my kit (SMART RACE for BD-
It will help if you use gene specific primer for cDNA synthesis. For RACE, use another GSP that is 5' upstream the first primer.
Okay, but how do I combine the specific primer with the kit, do I just add it in the reaction mix? At what concentration, and should it be shorter than my RACE primers (28 nt, Tm=72 degrees)? I gather I have to use the oligo and primer that comes with the kit too.
I see the problem now. I suppose that you are using a kit with adaptor-linked cDNA. In that case, you can not make your cDNA using GSP and use the cDNA for RACE. Sometimes RACE is hard which may not necessarily have anything to do with low copy number of transcripts. Try to find a source cell that has high expression of your gene.
Of course you can use a gene specific primer for your first strand synthesis. Just replace the 5┤-CDS Primer (from the BD Kit) with your gene specific primer (12 ÁM). The first race PCR should be with a nested gene specific primer (use touchdown PCR to enhance specifity) and with the nested universal primer (kit). If you see no product or a smear, do another nested PCR with your PCR product. Sometimes it is hard to optimize the PCR conditions but it should work.