Rnase Protection Assays

Pamela Norton pnorton at lac.jci.tju.edu
Tue Sep 12 15:18:25 EST 1995


In article <voeller-1009952343080001 at voeller.his.com>, voeller at his.com
(Jim Voeller) wrote:

> In article <42pdvg$dal at apopi.u-strasbg.fr>, Francois Nantel
> <Nantel at titus.u-strasbg.fr> wrote:
> 
> > I've never done and never needed to. I usually either precipitate the
> > probe or purify it on a spun column.
> > 
> > Francois
> 
> Yes I agree. I have done hundreds of RNAse protection assays without ever
> gel purifying the probe. If you feel that your probe is not full length or
> is somewhat degraded, then resynthesize the probe. Gel purifying seems
> like a real pain and could lead to the degradation that you are trying to
> avoid.
> Jim Voeller
> Georgetown University, Washington DC 
> voellerj at gunet.georgetown.edu

    As I believe someone else pointed out, this is somewhat
probe-specific. Some  templates yield specific, prematurely terminated
reaction products which can give unwanted extra bands later. Gel
purification eliminates these shorter products, and completely removes
unincorporated nucleotide (not critical for RNase protection, but
important for other applications). I have _never_ experienced degradation
of the transcript during gel purification (at other steps, yes). However,
I admit that it is an extra manipulation. A safe suggestion is to try
without gel purification, resorting to gels only if extraneous bands are a
problem.

-- 
Pamela A. Norton, Ph.D.          Assistant Professor of Medicine
Thomas Jefferson University
Philadelphia, PA 19107           p_norton at lac.jci.tju.edu



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