5' RACE

Steve Scherer sscherer at helix.nih.gov
Sun Mar 3 16:12:09 EST 1996


I just finished using the Marathon-Ready cDNA from Clontech (a rat brain
cDNA library with fancy adaptors ligated to the ends). You'll need to
generate a few gene-specific primers and try all of them both individually
or in combinations as nested primers where appropriate. I recommend using
touchdown PCR and then blotting and screening an aliquot of your PCR
reactions to find appropriate bands or smears to cut out and gel purify
before cloning and sequencing. A word of caution though: I found that
whoever constructed this particular library at Clontech didn't do a very
good jump of removing the original cDNA primers and found a number of
clones containing at least parts of this sequence ligated between the
adaptor and the 5' end of the message. Other than that, a fine system.
Hope this helps - email me if you have any other questions.

Happy sequencing,
Steve
sscherer at helix.nih.gov

In article <4h77iq$7dh at tribune.usask.ca>, bonhamp at duke.usask.ca (Peta C.
Bonham-Smith) wrote:

> I need to carry out some 5' RACE to locate the 5' end of a message of 
> interest - do I need Poly A+ RNA for this procedure?  If so what is the 
> recommended procedure these days - I really don't want to go back to 
> oligo-dT columns!!!  Secondly, which kit or procedure could you recommend 
> for the 5' RACE itself?
> 
> Please reply to
> bonhamp at duke.usask.ca
> 
> Thank you
> 
> Peta Bonham-Smith
> Department of Biology
> University of Saskatchewan
> Saskatoon
> Canada



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