> Ken Howe wrote:
> Dear Netters,
> What's the concensus on DEPC-treating buffers (PIPES, HEPES, Tris,
etc) > or salt solutions (NaCl, MgSO4, MgCl2, Mn++, Zn++ etc) vs
> sterilization vs autoclaving for removal of RNases? Can I treat
> previously made solutions, or do I need to make them in DEPC-treated
> water over again?
> Eric Anderson replied -
> one thing to keep in mind is that you can't treat any buffer with a
> free amino group (i.e. Tris) with DEPC...best thing that you can do
> is sterile filter or autoclave and hope for the best.
It's also not a good idea to autoclave buffers like Tris: we prepare
stocks of DEPC-treated water and use them to make up Tris and other
solutions for RNA work. Part of the treatment is an autoclaving step
anyway. If you're really paranoid you can filter sterilise the final
solutions, but we normally don't and have had no problems so far. I
suspect that RNases would pass through a 0.2 micron filter anyway, but
it would keep the bugs out.
An important point about DEPC treatment of water is to stir thoroughly
after autoclaving to drive off residual ethanol. We normally stir
vigorously for at least 2 hours on a magnetic stirrer.
Paul Hand Horticulture Research International
Handp at bbsrc.ac.uk