How do you prepare RNase-free solutions for Northern blotting?

Hi,

How do you prepare RNase-free solutions for Northern blotting? For
example, I want to prepare RNase-free 20X SSC or solutions containing
SDS. I planned on using 0.1 % DEPC to make the solutions RNase-free
but I wonder whether DEPC may react with any of the components. And
since autoclaving is necessary for deactivating DEPC, can the
solutions be autoclaved without degradation of any components?

Paul

Paul Wary wrote:
Anything that can be autoclaved and does not contain amine groups (such as
Tris) can be treated with DEPC and autoclaved. Anything that cannot be
autoclaved (some metal salts, etc) or contains amine groups can be made in
baked glassware with DEPC-treated water and dedicated chemical stocks (ie.
no spatulas, no replacing overweighed amounts).

By the way, if you're only planning on using the 20X SSC for transfer to the
nylon membrane, you don't have to worry about making it RNAse free. The salt
concentration is too high to support RNAse activity.

On 12 Nov 2004 08:25:10 -0800, [Only registered users see links. ] (Paul Wary) wrote:


I gave using DEPC many years ago, and my RNA looks fine. I do use very
good water (18 mega-ohm, low TOC, etc., as in MilliQ or equivalent),
however.

Nick

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Nick Theodorakis
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Paul Wary wrote:

Besides what said by Kyle Legate that is certaintily right (like the
reply to a similar my question of 01 november) I can add thar I have
recently read on the Ambion catalog of an alternative reagent to the
DEPC, that can be used also with Tris (RNAsecure...) and of a solution
that decontaminate the electrodes of pH meter (Elecrozap) (that usualy
I don't know how to decontaminate).
Anyway I never used this products so i can't say if they are realy good
and advantageous or not.
Bye.

Fabio