Help with RNA extraction method

George Rutherford gruther at bilbo.bio.purdue.edu
Fri Aug 9 15:40:38 EST 1996


In article <mantei-0708960901590001 at retrograde.ethz.ch>,
mantei at neuro.biol.ethz.ch (Ned Mantei) wrote:

> In article <4u8tiq$gvg at enyo.uwa.edu.au>, karenmk at uwa.edu.au (Karen
> Kroeger) wrote:
>  The RNA is degrading when I incubate at 65C in Promega's PE buffer
> > (primer extension kit : Tris, KCl, MgCl2, DTT, spermidine, ph 8.5).
> 
> Probably alkaline hydrolysis--pH 8.5 is already alkaline enough to be
> unhealthy for RNA over the long term, and incubation at 65 C makes the
> reaction go much faster. Cleaning up the RNA would in this case not help.
> 
> -- 
> Ned Mantei
> Dept. of Neurobiology, Swiss Federal Institute of Technology
> CH-8093 Zurich, Switzerland
> mantei at neuro.biol.ethz.ch   Fax: +41-1-633-1046

I doubt that alkaline hydrolysis is the problem as the pH of Tris buffers
decreases approximately 0.03 units per degree of temperature increase
which would place the reaction pH at about 7.3. That should be OK. I
assume that you have checked the quality of the RNA on a gel and it's OK
before going into the buffer? Sounds like a tough one.

George



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