Please share experiences of using DEPC reated water?
David F. Spencer
dspencer at is.dal.ca
Tue Mar 13 17:05:29 EST 2001
<45524439D771D311BF3E0090274F0456023B893E at wpg001ex6.gov.mb.ca>,
AHamel at agr.gov.mb.ca ("Hamel, Andre (AGR)") wrote:
> Several colleagues and I have each been working with RNA and DNA since
> 1982 and in the past have often used DEPC treated distilled water for all
> molecular biology protocols. After a few years (1985 - 1990) several of
> us had become suspicious about the value of using DEPC (0.1% v/v) double
> distilled water in relation to how stable RNA, DNA and enzymes are in
> DEPC treated, autoclaved water. We wondered that if the DEPC had not been
> completely removed by autoclaving and/or too much had been added to the
> water (noticed by both smell after autoclaving and storage on bench in
> loosely opened sterile bottles for months and/or the water foams even
> very slightly when shaken), then problems often "creep up" apparently as
> result of using DEPC treated water.
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.
> Our labs have always had minimal to no problems obtaining quality
> purified RNA/DNA (we all use robust GiTC/phenol/CHCl3 extractions along
> with vigilent sterile technique, gloves, clean equipment, supplies, etc).
> However, our suspicions had been confirmed on several occassions when we
> had actually tested to compare the effects of using fresh distilled water
> compared to the same distilled water which had been "properly" treated
> with DEPC (0.1% v/v DEPC, sat on bench overnight then autoclaved for 30 min).
> We suspected that despite autoclaving DEPC treated water, far too often
> (although not always) reactions and stability of RNA/DNA could at times
> be severly compromised.
You essentially answered your own question about whether DEPC treatment
makes any sense. I've worked with RNA for over 20 years (and a colleague
for about the same time) and we have never used DEPC for anything other
than chemical sequencing of nucleic acids, which in my opinion is its
only real use. The smell is fairly pleasant though, I must admit.
If RNases cause problems during RNA isolation they are either coming
from the tissue that's being extracted or the people during the work are
careless and/or work in dirty lab conditions. A water purification
system that has RNases needs to be repaired or replaced; the
contaminating RNases will be the least of its problems.
David F. Spencer, PhD
Dept. of Biochemistry and Molecular Biology
Halifax, Nova Scotia, Canada
dspencer at is.dal.ca
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