Please share experiences of using DEPC reated water?
Rimantas.Plaipa at gf.vu.lt
Fri Mar 23 09:55:05 EST 2001
"David F. Spencer" wrote:
> In article <3AAFB214.F3F9FB31 at voruta.vu.lt>, Rimantas.Plaipa at gf.vu.lt
> (Rimas) wrote:
> > 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.
Yes, such was my initial idea which started my interest in these things.
Water from air absorbed by DEPC may create some amount of ethanol, which
causes breakdown of DEPC into DEC and CO2. This reaction doesn't consume
neither water nor ethanol - ethanol acts as pure a catalyst, so it may
lead to quite substantial degradation event if only minute quantities of
ethanol(or water) are present.
My own investigations and data from literature about kinetics of
hydrolysis of DEPC in water (half time <30 min) asserts that no DEPC can
be present in water after 24 h (if we believe in chemical kinetics and
exponents). So I aranged gas-chromatographic - mass-spectrometer
analysis of such smelling solutions and the only compound found was DEC.
> 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.
Well, DEC is probably persent in small amounts in DEPC only(few percents
maybe) so it may remain disolved in water. Esters hydrolyse easely in
alkaline or acidic media but this doesn't mean that they are very
unstable in neutral medium. The second order constant of alkaline
hydrolysis of ethylacetate is ~0.1 M-1*s-1 which translates into 10-8
s-1 at pH7 (half time of several years). I don't have data about DEC but
it should be more stable than ethylacetate.
> 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.
After gas-chromatographic studies of DEPC-treated solutions I tried to
detect DEC in stock DEPC. Gas chromatography cannot be used because at
high temperature DEPC will breakdown and produce DEC anyway. So I tried
liquid chromatography and ordered the real DEC (from Sigma) to be used
as a standard. These experiments weren't succesful because the
absorbtion of UV by DEC is so low (and absortion of many other compounds
so high) that I couldn't even identify its peak in chromatograms of that
commercial DEC (and I didn't have any other useful detector). But at
least I was able to check its smell and it's identical to that of DEPC.
And because of better volatility (boiling pt 126 C vs 180 C for DEPC)
its smell should be sensed at much lower concentrations (haven't tried
to check it experimentally though).
> 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.
The reasons are not totaly foolish - DEPC probably would be good if
indeed were RNases in the solutions. Simply it's not necessary because
there are no RNases in water and stock reagents unless someone spits
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