Why MOPS for RNA gels?

[Glen Shearer]

Just out of curiosity....why does everyone use MOPS
buffer for RNA gels? Why not use autoclaved TBE or TAE?

Also, I recall a note in Biotechniques about leaving out
the formaldehyde in the gel. The idea was that the
formamide/formaldehyde in the sample buffer denatured the RNA
and it separated fine! Anybody ever try this?

Glen

[Tracy Aquilla]

In Article <371sh0$8...@server.st.usm.edu>, gshe...@whale.st.usm.edu (Glen

I think MOPS is used because it's traditional. I suppose it's a better
buffer than Tris though. For a good reference on running gels without
formaldehyde, see NAR 21(11): 2783-4.

Tracy Aquilla, Ph.D.
Molecular Physiology and Biophysics
University of Vermont
aqu...@salus.med.uvm.edu

[NICHOLAS THEODORAKIS]

In article <aquilla.1...@sadye.emba.uvm.edu>,

Tracy Aquilla <aqu...@salus.med.uvm.edu> wrote:
>In Article <371sh0$8...@server.st.usm.edu>, gshe...@whale.st.usm.edu (Glen
>Shearer) wrote:
>>

<snip>

>>Just out of curiosity....why does everyone use MOPS
>>buffer for RNA gels? Why not use autoclaved TBE or TAE?

<snip>

Think about the chemistry. How does formaldehyde denature RNA?
Aldehydes react with primary amines. Which is why formaldehyde or other
aldehydes are also used to fix samples for processing for microscopy
(cross-links proteins). So that's why you can't use it with Tris.

Nick

P.S. Not to waste band-width or anything, but we are now the proud
parents of twin baby girls: Catherine Marie, born Oct. 6, 6:51 a.m at 4
lbs., 10 oz., and Juliana Pauline, born Oct. 6, 6:52 a.m., at 5 lb. 3
oz. They are both healthy, beautiful, and 100% RNase-free (all that
placental ribonuclease inhibitor, I suppose :-) ).

Nick (aka "Daddy")

--
++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++
Nick Theodorakis
nt...@welchlink.welch.jhu.edu
Johns Hopkins Medical School, Baltimore, MD

[David S. Huen]

In article <371sh0$8...@server.st.usm.edu>, gshe...@whale.st.usm.edu (Glen
Shearer) wrote:

>
> Just out of curiosity....why does everyone use MOPS
> buffer for RNA gels? Why not use autoclaved TBE or TAE?
>

Could it be because Tris has free amino groups that will form Schiff
adducts with formaldehyde whereas the morpholine core of MOPS is
non-reactive to this? Its been a long time since I did my organic chem
though!!!!

--
David S. Huen Phone: (0223) 333921
CRC Human Cancer Genetics Group Fax: (0223) 333875
Dept. of Pathology e-mail: smh...@cus.cam.ac.uk
University of Cambridge
CB2 1QP
United Kingdom

[Jim Astwood]

In article <371sh0$8...@server.st.usm.edu>, gshe...@whale.st.usm.edu (Glen
Shearer) wrote:

I find that lowering the percentage of formaldehyde in the gel to around 3%
has no deleterious effects, and makes the gel considerably less nausiating.
Some people add formaldehyde to the tank buffer too, but this is not
needed at all.

Jim
--
James D. Astwoood
Monsanto Company
700 Chesterfield Parkway N.
Mail Zone GG4K
St. Louis. MO
E-Mail: jda...@monsanto.com

[Keith Hutchison]

In article <darreng.1...@ccmar.csiro.au>, dar...@ccmar.csiro.au

(D.Greeve <DAEMON>) says:
>
>>In Article <371sh0$8...@server.st.usm.edu>, gshe...@whale.st.usm.edu (Glen
>>Shearer) wrote:
>>>
>>>Just out of curiosity....why does everyone use MOPS
>>>buffer for RNA gels? Why not use autoclaved TBE or TAE?
>>>
>

>MOPS is certainly a better buffer. I tried Tris once and it couldn't maintain
>the pH of the gel, so I got a very strange physical deformation of the gel and
>the Bromophenol Blue went Green....
>
>D.Greeve
>Dept. Animal Production.
>C.S.I.R.O.
>Western Australia.

Sorry if what I have to say has been said before. I just ran into this
particular thread and may have missed some earlier comments. It raises
one of my pet peeves though. pKa of Tris is around 8.1, which means
that Tris buffers in the low 7's (of which there are many) are greatly
reduced in their buffering capacity (one log for each pH unit). So,
yes, Tris electrophoresis buffers, when below pH8 are not as stable.
And with RNA one has to worry about the pH going too alkaline because of
the degradation problems. Ergo, MOPS, with a pKa of 7.2. Standard MOPS
gels are at pH7. This doesn't mean you couldn't get away with it,
particularly if your RNA is relatively abundant. Just a bit more risky.

Keith Hutchison
Biochem, Microbiol and Molec Biol
UMaine

[Oleh Kevalchuke]

Keith Hutchison (KEI...@MAINE.MAINE.EDU) wrote:
: In article <darreng.1...@ccmar.csiro.au>, dar...@ccmar.csiro.au

I run 18S rRNA on agarose gel buffered with TBE with no problems.
I added 4 M urea to the sample.

[D.Greeve <DAEMON>]

>In Article <371sh0$8...@server.st.usm.edu>, gshe...@whale.st.usm.edu (Glen
>Shearer) wrote:
>>
>>Just out of curiosity....why does everyone use MOPS
>>buffer for RNA gels? Why not use autoclaved TBE or TAE?
>>
>>Also, I recall a note in Biotechniques about leaving out
>>the formaldehyde in the gel. The idea was that the
>>formamide/formaldehyde in the sample buffer denatured the RNA
>>and it separated fine! Anybody ever try this?

>I think MOPS is used because it's traditional. I suppose it's a better

>buffer than Tris though. For a good reference on running gels without
>formaldehyde, see NAR 21(11): 2783-4.

MOPS is certainly a better buffer. I tried Tris once and it couldn't maintain

[Malcolm Moos Jr., M.D., Ph.D.]

Tris is a primary amine, so you can't treat it with DEPC. The
formaldehyde can be reduced (protocol in Farrell's book) with no
problem.

Malcolm Moos Jr., M.D., Ph.D.