Please share experiences of using DEPC reated water?
David F. Spencer
dspencer at is.dal.ca
Thu Mar 22 16:13:53 EST 2001
In article <3AAFB214.F3F9FB31 at voruta.vu.lt>, Rimantas.Plaipa at gf.vu.lt
> "David F. Spencer" wrote:
> > Although DEPC is considered to be unstable in water solution it does
> > persist at low levels. I DEPC-treated one of my stocks of BLOTTO years
> > ago, immediately after placed it at 65C for considerable time to try to
> > break down the DEPC, and the solution still has that sweet DEPC smell
> > when you take off the cap and sniff it. And this has been sitting in a
> > solution of 5% skim milk powder! People who don't see negative effects
> > of working with DEPC-treated water may be saved by using Tris (or
> > similar chemicals) which react with DEPC and so scavenge the residual
> > DEPC from the original water.
> No, DEPC doesn't persist. The smell which remains in DEPC treated
> solutions belongs to diethylcarbonate (DEC) which is present in small
> amount in commercial DEPC and also may accumulate during storage due to
> spontaneous degradation of DEPC (decarboxylation). DEC is pretty inert
> and therefore should not ihibit enzymes (well, theoretically).
That is an interesting proposal on which I'll comment.
I am not familiar with the synthesis chemistry of DEPC so it is indeed
possible that commercial DEPC has DEC impurities.
However, it appears (cf. Ehrenberg et al.) that the breakdown of DEPC
via DEC requires ethanol/methanol and/or water as catalyst. Ethanol is
of course a final breakdown product of DEPC so it could accumulate from
that reaction, presumably catalyzed by water vapour from the air being
absorbed in the original DEPC stock. To generate ethanol some DEC must
hydrolyze and that final step would require water to be present.
In my final reaction mix (skim milk powder plus SDS, pH 7.9) the
starting quantity of DEPC was above saturation (not too difficult given
DEPC's poor water solubility) but final ethanol concentrations from DEPC
hydrolysis could never exceed maybe one or two percent. And although DEC
is essentially insoluble in water it clearly must have a water boundary
of some sort. Given the ease of hydrolysis of esters in water I find it
hard to believe that DEC could persist for years even in what would
start as a biphasic water mix.
The residual smell of my old BLOTTO solution seems more like DEPC than
an ester (that's pretty subjective of course). I admit that I have
always found it hard to believe that DEPC could persist for so long and
it is possible that the smell from my BLOTTO is neither DEPC or DEC but
some other reaction product.
Regardless, this doesn't affect my original point that DEPC has become
to many molecular biologists a talisman to protect against the RNase
gods. The reasons for most of its use are foolish and poorly thought out
and I would bet that hanging a clove of garlic around your neck during
RNA preps would be no less effective than obsessively DEPC-treating
anything and everything used during RNA preps. And there will be
situations where DEPC treatment is not innocuous.
David F. Spencer, PhD
Dept. of Biochemistry and Molecular Biology
Halifax, Nova Scotia, Canada
dspencer at is.dal.ca
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