Rapid Amplification of cDNA Ends (RACE)
pmiguel at bilbo.bio.purdue.edu
Mon Sep 23 10:23:13 EST 1996
3' RACE works okay with an oligo-T primed reverse transcription --
but I never had any luck using just the oligo-T as a PCR primer. Instead
I used an oligo-T+adaptor primer where the adaptor was 20 bases of some
reasonable G+C content containing a couple of restriction site for subsequent
cloning of the band. The PCR I did with just the adaptor (no oligo-T; i.e.
another primer) and the gene-specific primer.
Even so I had to do 2 sets of 40 cycle PCR rxns -- the second set with
a nested gene-specific primer. I never could trust the bands I got as
being the real thing -- I always used a Southern to check out which bands
were a specific product. I think this may be an organism-specific problem.
I did my amplification from maize endosperm mRNA. Most, if not all, maize
RNA contains lots of LTR-retrotransposon RNA in it. I doubt this is true
of many organisms.
Also, products larger than 500 bases were very weak. I don't think I ever
saw a product larger than 700 bases without hybridization. PCR techniques
are more sophisticated now. So this may not be a great problem.
In article <51vdfa$c8i at pulp.ucs.ualberta.ca>, plagali at gpu2.srv.ualberta.ca
(Pamela Lagali) wrote:
> I have been attempting to look for full-length cDNAs using both 5' and 3'
> RACE, however have had no success with the 3' RACE procedures I have
> tried. I think the problem may lie in the steps subsequent to
> first-strand cDNA synthesis with a gene specific primer, namely PCR using
> an oligo(dT)-based reverse primer. Control PCR reactions using two
> gene-specific primers have worked, but I have been unable to obtain
> further downstream sequences closer to the 3' poly(A)+ tail. Does anyone
> have any experience with RACE? Has anyone experienced success with PCR
> using an oligo(dt)-based primer to amplify 3'-end cDNA sequences?
> Any assistance would be greatly appreciated!
> Pamela Lagali
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