Rapid Amplification of cDNA Ends (RACE)

vilimf01 at mcrcr6.med.nyu.edu vilimf01 at mcrcr6.med.nyu.edu
Tue Sep 24 23:09:54 EST 1996


You need to do the reverse transcription using an OdT18 + anchor
sequence (any sequence will do - like T7 promoter for example) in
order to get a reasonable annealing temperature. The other alternative
is to use a 30-35mer oligodT which will have an annealing temp of
60-70 C (2xT).  I have used the anchored 3' RACE sucessfully, but PCR
from libraries is easier(if you can find an appropriate library), just
by using vector specific primers.  Southern screening of PCR products
with an internal gene specific primer is a must (IMHO) to identify the
correct bands.  High annealing temps are also useful (like stepdown
PCR) to get good yields of correct products.  In my experience, RACE
always gives multiple bands.  I would also stay away from nesting, I
have gotten burned before by extraneous crap sequences adding on in
nesting.  Hope this helps

				Usual Disclaimers		SVEN


In article <51vdfa$c8i at pulp.ucs.ualberta.ca>, plagali at gpu2.srv.ualberta.ca (Pamela Lagali) writes:
> Hi,
> 
> 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!
> 
> Thanks,
> 
> 
> Pamela Lagali
> Department of Cell Biology
> University of Alberta
> Edmonton, Alberta, Canada
> T6G 2H7



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