RT-PCR question

J. Carvajal jcarvaja at hgmp.mrc.ac.uk
Fri Sep 27 11:46:38 EST 1996


Looking for some ideas.

I am trying to amplify an alternatively spliced gene from mRNA.  The size
of the expected products ranges from 1.2-7.5 kb in size.  I do have the
3'UTR and the 5'UTR but I do not know the full exonic distribution in
between.

I have been trying several enzymes (long range, Taq2000, normal Taq) and
different PCR conditions, but I only obtain amplification of products up
to 2kb, while the larger ones do not amplify at all.  I think that the
expression levels of the larger mRNAs is very low and, if we add the
preferential amplification of the smaller products you will have an idea
of my problem.

Any suggestions?  Any ideas?

All welcome.

Thanks in advance,

J.J.Carvajal





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