Why DTT in RNA prep?

Robert Hartley rh at mblab.gla.ac.uk
Tue Sep 14 10:42:38 EST 1999


Peter wrote:
> 
> In article <37DE1B53.9C988AFE at uni-tuebingen.de>, Byung-Hoon Kim
> <byung-hoon.kim at uni-tuebingen.de> wrote:
> > Does anyone have any idea why reducing agent has to be in lysis 
> >buffer for RNA prep?
> > Thanks in advance for any comments.

> A final concentration of 1 mM DTT at pH of 8 should be quite effective at
> reducing all proteins.

Yep it is to potentially inactivate RNases. 
BUT!!! ensure your DTT is pH 8+ the reason is:- DTT is 10x more
effective at this pH compared to pH 7.0(ish). I think the ref is
clelland/McClelland about 1987(ish). I can dig it out for you if you
mail me.

Another thing is use a fresh DTT solution. I have no experiance of old
DTT soln's but Bob Klebe who developed a RT-PCR method without RNA
isolation said that old DTT was the primary reason for problems with his
method.

Cheers
Bob



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