Please share experiences of using DEPC reated water?

Michael L. Sullivan mlsulliv at facstaff.wisc.edu
Fri Mar 2 16:19:05 EST 2001


I can't point to any literature, I can only relate my experience.  I have
worked in labs where we used DEPC religiously, and in labs where we didn't
use it at all.  From everything I have heard in this newsgroup, very often
a "good" water source is RNase and DNase free already (e.g. very often
milliQ water right out of the tap works fine).

However, when I was in a lab where we used DEPC extensively (and I mean
extensively-- we DEPC treated 10 l carboys of SSC without autoclaving), I
never saw any of the problems you described.  For example, I used to always
resuspend RNA in DEPC treated water.  Once, somebody knocked a box
containing my samples out of the -80 freezer.  They were at room
temperature somewhere between overnight and 2 weeks. Amazingly, I could
detect no degradation fo the RNA at all.  Also, all of my RNA samples were
still good when I left that lab, certainly many were over 2 years old.
Also, nobody every seemed to have any trouble with enzymatic reactions.
Frankly, I think DEPC is far too unstable in the presence of moisture to be
causing any of the effects you describe.

However, it has been noted several times in this newsgroup that autoclaving
can be very problematic for introducing RNAses into supposedly clean
materials via aerosols (e.g. from autoclaving media with yeast extract, or
waste materials).  These would seem to be potentially far more of a problem
than the DEPC itself, although I do routinely autoclave materials and
reagents for RNA work... I've never experienced the problem.

This is just my experience.

Mike





>  My question is, can anyone recommend any literature (BRL Focus, other
>journals) regarding the pros and cons of using DEPC treated water for
>molecular biology work?  And what is everyone elses experience with this
>apparent dilemma. So many commercial kits, protocols, lab manuals, etc
>recommend using DEPC treated water for nearly everything. Please post
>replies. I will post any emails I receive. We used to use double glass
>distilled water, sit overnight with 0.1% v/v DEPC, then autoclaved 30
>minutes in 100 mL and 500 mL CLEAN glass bottles (which had been soaked for
>several minutes chromerge-chromic-culfuric acid washed, rinsed well ... talk
>about paranoia, eh? ... this back in days when GiTC methods were still quite
>new  8']
>   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.  "Problems" include what appears to be
>degrading of RNA and DNA after prolonged storage at -70*C (>2 weeks),
>efficiency of reactions decreasing over time (after going back to same
>RNA/DNA samples which previously worked fantastic when first tested, only to
>not work as well or not at all a few weeks after storage at -70*C, some
>appear to just "die" ... reactions such as ligations, PCR, in vitro
>translation, in vitro transcription, etc.
>   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.
>
>Thank you,
>
>Andre Hamel
>
>
>
>
>---


Michael L. Sullivan, Ph.D

U.S. Dairy Forage Research Center
1925 Linden Drive West
Madison WI, 53706

(608) 264-5144 Phone
(608) 264-5147 Fax


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