"Re: Yellow MOPS - why"-- have we beaten this dead horse enough?

Michael L. Sullivan mlsulliv at facstaff.wisc.edu
Thu Mar 23 12:08:33 EST 2000


Well, I'm gonna' take my turn beating this dead horse!

>
>-I am not sure I understand this one?
>-RNases do get destroyed by sufficient autoclaving (30min). Otherwise I would
>-not have been able to see any RNA in my gels, since I do not use DEPC.
>-And what has LB got to do with the isolation of RNA?
>
>RNAses are heat resistant: you can autoclave for hours if you want to,
>they won't be destroyed. That makes sense too; because in the living cell
>the RNAses have to be very stable to remove the mRNA which is not needed
>anymore (rapid turnover).

It seems to me that neither of these overly general statements is correct,
since RNAses are a somewhat heterogeneous group of enzymes carrying out
lots of different functions in cells.  My guess is that there are RNases
that are very labile, and others that are relatively stable.  I've been
told by people who work on A-type RNAses that even those are not as stable
as most people believe (but these are also so active that even if
autoclaving destroyed 99% of their activity, the remaining 1% might be
enough to cause a problem in some instances).  It's probably safe to say
that autoclaving would reduce RNAse activity in a solution, but there would
be no guarantee that the solution would be free from contaminating RNAse
activity.  I'd also venture to say that for most people, the autoclave is
not a likely source of RNAse contamination, and in fact probably does help
some.

I'm also not sure that in vitro stability has all that much to do with in
vivo stability and/or in vivo function.

I personally think that conditions in different labs are highly variable so
what a researcher has to do to have a sufficiently RNase free environment
ends up being somewhat empirical.  For example, I never bake or DEPC treat
anything, and use lots of autoclaved materials, and have rarely had a
problem (I remember one instance in 8 years that was eventually traced to a
buffer somebody else made).  In some labs, this approach wouldn't work
though, perhaps due to a less good water supply or some other source of
contamination that might be hard to track down and control.  I say err on
the side of caution with your precious samples, but maybe see what you can
get away with on easily repeated experiments!

Hope I didn't ruffle any feathers here, but that's my experience!

Mike



Michael L. Sullivan, Ph.D

Institute for Molecular Virology
University of Wisconsin-Madison
1525 Linden Drive
Madison WI, 53706

(608) 262-3035 Phone
(608) 265-9214 FAX


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