Molecular cloning -Sambrook et al/ Tris base vs TRIZMA

Stephen R. Lasky, Ph.D. Stephen_Lasky at brown.edu
Tue Jun 6 18:35:08 EST 1995


In article <pnorton-060695105705 at norton1.jmc.tju.edu>,
pnorton at lac.jci.tju.edu (Pamela Norton) wrote:


>                  YES!!!!!! 
> 
>     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,
> 
>                                                                                                        
Pam Norton
> 

Pam:  No!!!!!   8-)   Tris base and trizma are the same things.  Look them
up in your Merck index.Trizma is just sigma's trade name for tris base
(you'll notice the registered trademark mark on sigma's trizma): 
trishydroxymethylaminomethane.  MW=121.1, mp=171 to 172 for both tris and
trizma.  I have been using trizma or tris interchangably for 20 years now
and never could ascribe the problems you cite with your gels to using tris
or trizma (I am using the molebiol grade from trizma from sigma as well as
tris from gibco/brl). .  Empirically, you may have solved your problems
when you changed from Trizma to tris, but I would suspect a pHing problem
or bad water (especially if the problem was at RWMC), or lower quality
trizma/tris.

On the original question, I only have the old version of Sambrook (circa
1982) and it just says to use TrisHCl pH6.9 for the stacker and TrisHCl pH
8.7 for the running gel. In the appendix it says to pH the Tris Base with
HCl. We always just dissolved the tris in DI-H20,  pH'd it to whatever we
wanted with HCl (at the temp that we were going to use it or taking into
acct the change in pH with temp) and then brought it up to the final
volume needed.  But, if you look in the ISCO tables (something that you
needed on your desk if you were studying biochem back in the 70's) you can
see that they give formulas for making 0.5 M tris at any pH by mixing Tris
base with Tris HCl, so you can do it that way also.  It probably doesn't
make any difference which way you get Tris-HCl to the pH you want, as long
as you have the right molarity and pH.

On the Cl- thing, There are two questions here, one on buffering and one
on disc electrophoresis.  Seems to me that it is the H+ that is impt for
the tris buffer.  Buffers are there to resist pH changes.  The way they do
that is by sucking up H+ ions (H30) effectively tying them up and keeping
them out of the solution.  Buffers work best at their pK where they are
half protonated and half deprotonated, that way they can either give up or
suck up a proton when [H3O] changes in the system.  The Cl- is the counter
ion since you really can't get a bottle of H+. The counterion is important
for disc electrophoresis as it is the basis for  the stacking effect
(along with glycine).  A guy named Kohlraush (something like that) back at
the turn of the century (I think), came up the a normalizing function
describing the behavior of ions in an eletrical field that was the basis
for discontinuous gel electrophoresis (which was introduced in the early
50's).  There have been some excellent discussions in this group by people
who can explain that phenomon better than I, so if you are interested in
the theory I will dig up their addresses.

If I'm wrong about the above, please let me know but I hope that helps.

SRlasky

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Stephen R. Lasky Ph.D.  Brown University/Roger Williams Medical Center
Landline: 401-456-6572   Fax: 401-456-6569  E-Mail: Stephen_Lasky at brown.edu
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