RT-PCR in GC-rich Chlamy

Julie Knott jknott at biosci.cbs.umn.edu
Wed Dec 18 07:55:09 EST 1996


I am looking for advice from anyone doing RT reactions on our GC-rich
Chlamy!! I have performed RT reactions with both random hexamers and gene
specific primers using Superscript II RT (GibcoBRL) with successful
results in all but one region.  I have complete genomic sequence in
this region and can somewhat predict the cDNA sequence, (the RT-PCR will
confirm it).  The problem region appears to have at least one 40bp region
of 82% GC!  I assume that the RT reaches that region and cannot proceed.

Has anyone tried performing RT reactions for the purpose of RT-PCR
or RACE on very high %GC templates?  What methods did you use to keep the
RNA denatured?

Thanks in advance,

Julie Knott (Porter Lab)
jknott at biosci.cbs.umn.edu

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