RT-PCR in GC-rich Chlamy

Thomas Thomas at hastingsmac2.fas.harvard.edu
Sat Dec 21 12:41:03 EST 1996


In article <598pjd$p9k at net.bio.net>, Julie Knott
<jknott at biosci.cbs.umn.edu> wrote:

> Hello!!
>
> 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

Try one of the DNA polymerases from Thermus thermophilus (available from
e.g. Promega, Boehringer, Perkin Elmer). RT works at 72oC which overcomes
the problem of the secondary structure.

Thomas

--

Thomas Urbig
Email:  Thomas at hastingslab.harvard.edu






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