Molecular cloning -Sambrook et al/ Tris base vs TRIZMA
pnorton at lac.jci.tju.edu
Tue Jun 6 10:57:05 EST 1995
In article <Pine.A126.96.36.1990605144847.56848B-100000 at paris>,
hroychow at NMSU.EDU (Hiranya Roychowdhury) wrote:
> On 5 Jun 1995, T.S. Pillay wrote:
> > In the electrophoresis section in Molecular Cloning by Sambrook et al
> > (a.k.a. the "Maniatis") it says not to use TRIZMA for SDS PAGE
> > electrophoresis buffers but Tris Base which should be pH'd to the
> > appropriate pH instead of using a mixture of Trisbase and Tris-HCl. Does
> > anyone know the reason for this.
> But I thought Tris base and Trizma were the same. The compound used for
> electrophoresis is tris(hydroxymethyl)aminomethane which, from Sigma,
> comes as TRIZMA (base). The TrisHCl is simply the hydrocloride of the
> above and is extremely acidic.
> I have never come across any protocol where the Tris buffer is
> prepared by mixing Tris base and hydrochloride to obtain a specific pH. I
> am not sure what the authors in Sambrook et al. meant, but I will not be
> surprised if it is a mistake. There are a few very obvious yet serious
> mistakes in that manual. The question is that whether, by using Trizma
> base, anybody has encountered any problem in SDS-PAGE!
TIn response to the above:
I wasted over a month trying to figure out what the heck had gone wrong
with my SDS-PAGE gels after setting up a new lab. Gels ran slowly, with odd
voltage/current ratios, bands were fuzzy, resolution was poor in a critical
mol. weight range (ca. 60 kDa). Problems were solved by formulating all the
buffers with Tris base (we get it from Gibco/BRL, no affiliation,
presumably other sources work as well) instead of Trizma. I too was
skeptical but tried this as a last resort based on the comment in Sambrook
et al. The difference was astounding.
However, I still don't know the correct answer to the original poster's
question, but I believe the Cl- ion concentration is the critical thing.
Hope this helps prevent someone from wasting their time,
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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