Autoclaved MOPS: is it supposed to be yellow?

[Brett Burkholder]

I'm trying to set up an RNA gel for a Northern blot.

The protocol I'm using, Current Protocols in Molecular Biology 4.9.1
-4.9.8, gives the recipe for MOPS buffer (10X: 0.4 M MOPS, 0.1 M Sodium
Acetate, 0.01 M EDTA).

I made the solution, added DEPC to treat for remaining RNases, then
autoclaved it. Now my MOPS buffer is yellow, so I'm wondering if
something is wrong.

Is this the correct way to make the buffer, or should I be making it
with water which has previously been treated with DEPC and autoclaved?

Brett Burkholder

[David L. Haviland, PhD]

On Sat, 28 Feb 1998, Brett Burkholder wrote:

> Date: Sat, 28 Feb 1998 02:58:30 +0000
> From: Brett Burkholder <bbu...@emory.edu>
> To: met...@net.bio.net
> Subject: Autoclaved MOPS: is it supposed to be yellow?

Brett:

In a word: Backwards...

Add mili-Q water to baked flask, add DEPC, stir with stirbar to mix.
DEPC won't go into the water but the idea is to expose as much of the
water to DEPC as you can. I usually let it stir for about 20-30 mins.

Autoclave water. Cool to RT.

Then make your MOPS solution per the recipe you have. Filter, we use
either 0.2 um or 0.45 um.

I would be dubious of the yellow MOPS. In fact, that is usually a sign to
discard it.

Hope this helps,
David

==========================
David L. Haviland, PhD
Assistant Professor, Immunology
University of Texas, HSC - Houston
Institute of Molecular Medicine
2121 W. Holcombe Blvd.
Houston, TX 77030
713.500.2413-Voice
713.500.2424-FAX
==========================

[Hiranya Roychowdhury]

At 02:58 AM 2/28/98 +0000, Brett Burkholder wrote:
>I'm trying to set up an RNA gel for a Northern blot.
>
>The protocol I'm using, Current Protocols in Molecular Biology 4.9.1
>-4.9.8, gives the recipe for MOPS buffer (10X: 0.4 M MOPS, 0.1 M Sodium
>Acetate, 0.01 M EDTA).
>
>I made the solution, added DEPC to treat for remaining RNases, then
>autoclaved it. Now my MOPS buffer is yellow, so I'm wondering if
>something is wrong.
>
>Is this the correct way to make the buffer, or should I be making it
>with water which has previously been treated with DEPC and autoclaved?
>

>Brett Burkholder
>
>

yes

Dr. Hiranya Sankar Roychowdhury
Plant Genetic Engineering Lab.
New Mexico State University
Las Cruces, NM 88003
Ph. (505) 646-5785
hroy...@nmsu.edu

[Deborah Hailstones]

I have never used DEPC treatment in mammalian RNA preps, seeing it as
one of those 'safety nets' that encourage slackness (like antibiotics
in TC....). I have found that if you are suitably meticulous and
cautious it's not necessary to use DEPC, and that way you can also
avoid exposing yourself to (yet another) toxin....
Having said that, my MOPS (in RNA-quality water, alone) ALWAYS goes
yellow after autoclaving and, if made up and then filtered rather than
autoclaved will eventually turn yellow with RT storage.. oxidation,
presumably.... yellow MOPS is definitely fine to use, no worries!

Deb

______________________________ Reply Separator _________________________________

Subject: Autoclaved MOPS: is it supposed to be yellow?

Author: <bbu...@emory.edu > at smtpgwy
Date: 2/28/98 2:58 AM

[J. Hendriks]

Brett Burkholder wrote: <34F77D...@emory.edu>...

>I'm trying to set up an RNA gel for a Northern blot.
>
>The protocol I'm using, Current Protocols in Molecular Biology 4.9.1
>-4.9.8, gives the recipe for MOPS buffer (10X: 0.4 M MOPS, 0.1 M Sodium
>Acetate, 0.01 M EDTA).
>
>I made the solution, added DEPC to treat for remaining RNases, then
>autoclaved it. Now my MOPS buffer is yellow, so I'm wondering if
>something is wrong.
>
>Is this the correct way to make the buffer, or should I be making it
>with water which has previously been treated with DEPC and autoclaved?
>
>Brett Burkholder

Nothing is wrong. MOPS-buffer is indead turning yellow during autoclaving of
the solution.
Brenda