HELP HELP HELP needed with RNA extraction
graham at biodec.wustl.edu
Sat Jun 8 12:04:17 EST 1996
> If you are merely autoclaving your glassware, it will not kill off all
> the RNAses. Baking of glassware is essential in most RNA preps. Also
> in some cases gloves won't do it either. Some need lab coat, hair net,
You can skip the hair net. Best to focus on the actual sources of
RNAses, which are biological samples including all "purified" enzymes,
particularily that relevant one used in plasmid isolation. :)
Anything which is not kept frozen and has had time to "grow"
on the bench is also a real problem, rather than a ghost story.
As Jean says, a great deal of effort which goes into avoiding RNAses is
probably unecessary, as they will not be found in distilled water,
properly cleaned glassware, new plastic, or on most people's fingers. If
you are spooked by the preponderance of such claims, test these
sources yourself with some RNA markers or rRNA!
As for DEPC, it has much greater potential for inhibiting your
subsequent enzymatic manipulations than protecting your RNA, in
my experience. Autoclaving will not remove all of it, unless perhaps you
leave it for many hours in the autoclave after the pressure come
down. If you water tests negative for RNAses anyway, DO NOT bother
with this dangerous chemical. I worked for seven years in an "RNA
lab", and DEPC never crossed the doorway. Make your buffers fresh from
powders, freeze them, and remake them often -same day at critical points.
Making sets of frozen aliquots which can be replaced as an entire group
when problems arise is the best of all.
You can fill your tip boxes by hand with gloves from fresh bags used only
by you -even if your fingers test RNAse negative, those of others may not.
Even more upsetting to some, we did not autoclave these materials and other
reagents for biochemical experiments at all, as this does not inactivate
RNAses, but does nicely release any that might be otherwise sequestered
inside of dust-borne microorganisms. Better to rely on freezing for
control of microorganisms in most cases, and filtration in the rest.
I'm always amazed by the differences of opinion on these things, as
anyone can easily determine the presence of RNAses quite simply
in their own supplies, buffers, water, and fingertips. Start your
project with RNAse assays, and dispense with the fear and DEPC. You
will breathe much easier, and be able to focus on the real sources
(largely endogenous, and a few introduced) of RNAses.
J. Graham PhD
Washington University of St. Louis
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