RNase contamination and yellow MOPS?!?
Dr. Peter Gegenheimer
PGegen at UKans.nolospamare.edu
Thu Mar 30 16:12:29 EST 2000
ð On 21 Mar 2000, Wolfgang Schechinger wrote:
ð -I'd appreciate any explanation why our MOPS buffer (pH 7, compsition
ð -below) got yellow after autoclaving. Is this commonly observed? .....
** As another poster said, MOPS isn't heat-stable. Fred Niedhardt's original
MOPS buffer for bacterial growth was to be sterile-filtered.
And if the yellow color were RNase, you'd be looking at several mg/ml protein
in your previously clear buffer!
On Thu, 23 Mar 2000 08:50:31, csc <un691cs at genius.embnet.dkfz-heidelberg.de>
ð RNAses remain fully active after autoclaving. And adding
ð DEPC won't help either, because the RNAses in the autoclave (for instance
ð from LB) may well diffuse into your bottle.
THe notion that RNase remain fully active after autoclaving comes from the
textbook example of RNase A refolding as a model for protein tertiary
structure. RNase A is held together by several disulfide bonds. When heated
and slow-cooled (for example, to inactivate contaminating DNases), native
RNase A will renature. When treated with GuHCl and dialyzed, RNase A will also
renature. However, when GuHCl-denatured in the presence of a reducing agent,
and then dialyzed w/o reductant, the enzyme remains inactive because its
disulfide bonds reform in the wrong combination.
>From this, one might conclude that autoclaving on liquid cycle (includes slow
cooling) will allow RNases to renature. However, in practice most labs that
work with RNAs under 1000 nt simply have no problems with RNase contamination
using water which is only autoclaved but not DEPC-treated. Macho mRNA people,
with 10 kb RNAs, may need more protection: it's a lab-specific issue.
Also, as other posters have said, there are a hundred sources of *potential*
RNase contamination. Don't forget that even "nuclease-free" BSA is often
contaminated! (This from personal experience.) Plain "5X recrystallized" BSA
is a rich source of nuclease. SO don't eat even the white snow...
| Dr. Peter Gegenheimer | Vox: 785-864-3939 FAX: 785-864-5321 |
| Department of | PGegen at UKans.nospam.edu |
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