RACE troubleshooting

[Peter Ashby]

In article <3C68266D.6803E5B0 at leland.stanford.edu>,
 Corinne RENIER <crenier at leland.stanford.edu> wrote:

> Hi,
> 
> Does anybody have experienced problems with the SMART RACE cDNA
> amplification kit (Clontech)?
> After the first strand synthesis, the RACE PCR using my gene specific
> primer (either 5' or 3') failed to give me clear band (I get smears),
> while my positive control (using 5'+3' gene specific primers) on the
> cDNA RACE populations (either 3' or 5' RACE cDNA population) is working
> under this PCR conditions (I've got a clear and nice band).
> I've designed different primers, but each time it's the same story, so I
> guess the primers are ok! I've been following the troubleshooting guide
> advices without success!
> Any advices or tricks to suggest? Does anybody have experienced with
> other RACE kits to share?

Have you tried nested pcr? It helps if your RT primer is a decent 
distance back from the edge of known sequence so you can design nested 
primers. In adition if there is a long enough lead in before known 
sequence you can pcr between the end of known and the start site to 
confirm the RT has worked and transcribed your gene. I made the previous 
incarnation of this kit work for 5 prime this way. Unfortunately I had a 
strong transcriptional stop and didn't get much more sequence. I could 
never refine down the multitude of bands I got trying to go 3 prime 
though.

Peter

-- 
Peter Ashby
Wellcome Trust Biocentre
University of Dundee, Scotland
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