5PrimeRACE-noway?

FABIAN MOEBIUS Fabian.Moebius at uibk.ac.at
Tue Mar 4 13:52:56 EST 1997


We desperately try to isolate the 5prime end of a clone. We already 
got 3prime partials. We also cloned the same cDNA from two other 
species with some difficulties (most but not all clones isolated from a oligodT 
primed cDNA library were incomplete). The problems could be due to a 
GC rich 5prime sequence.
We tried RACE with an oligodT primed library from 
Clontech). We do not get any 5prime end PCR products whereas 3prime 
PCR works. We now tried 5%DMSO which changed the picture with some of our 
antisense primers. 
Seems to be a trivial problem. Nevertheless we fail.
(1) Any suggestions how to overcome GCrich sequences in RACE?
(2) How could we increase the ratio between the desired longer (600bp) 
product and an undesired possibly truncated 300 bp amplicon?
(2) Any references for such problem (to make sure that it really is 
a problem)?
Thanks for your input!





Dr. Fabian F. MOEBIUS
Institut fuer Biochemische Pharmakologie
Peter Mayr Str. 1
6020 Innsbruck
AUSTRIA
TEL ++43-512-507-3155
FAX ++43-512-588627



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