Please share experiences of using DEPC reated water?

"Hamel, Andre AGR AHamel at agr.gov.mb.ca
Fri Mar 2 15:25:14 EST 2001


  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




---






More information about the Methods mailing list