DEPC treated water

[Priyanka]

How does DEPC react with water to inactivate RNAses and what is the optimum concentration..... When I used DEPC there were no bubbles that I could see... can Some one tell me how does DEPC behaves when one makes DEPC treated water..(right from pippeting step..)
Thanks

[Drosophilist]

Hi Priyanka,

Diethyl pyrocarbonate derivitizes histidine residues & inactivate nucleases including RNAse. DEPC is irritating to the eyes, skin, and mucous membranes, so be careful. Open the bottle in fume hood. Wear Gloves. It is a suspected carcinogen. It hydrolitically decomposes to CO2 & ethanol on heating.

We add 0.1 % DEPC (Sigma) to water in duran bottle. Close lid. Incubate bottle at 37degrees water bath overnight. DEPC would look like some transparent granules at the bottom of bottle. Next day autoclave with lid slightly loosened. DEPC would be gone.

Regards.
Sumit

[Priyanka]

Thanks Sumit!!!
I heared that when we pippete DEPC some effervescence comes???!!! ( I have no idea waht it means) I however did not get any of that.... Also i was not able to see any DEPC droplets when i added it to water....(There were many bubbles when i shook the bottle but they subside quickly) so we thought it has mixed quickly as well... And we are using a plastic botlle does that is any sort of a hinderance.......
Regards
Priyanka

[Jon Moulton]

Hi Priyanka,

DEPC can decompose, releasing carbon dioxide. That's the source of the effervescence.

The autoclaving step that Sumit mentioned is very important, as if DEPC contacts nucleic acids it can react with them and ruin their ability to base pair; this is likely the mechanism of carcinogenicity. Autoclaving decomposes the DEPC.

Henderson R.E.I, Kirkegaard L.H., &Leonard N.J. 1973. Reaction of diethylpyrocarbonate with nucleic acid components: adenosine-containing nucleotides and dinucleoside phosphates. Biochim. et Biophys. Acta, 294, 356-364.

It is possible that DEPC will react with some plastics. That reaction could compete with the reaction of DEPC with proteins, the latter being the reaction you want. While some plastics (e.g. polyethylene or polypropylene) would not react with DEPC, it is probably prudent to treat your water in a glass bottle to eliminate the possibility of reaction with the container.

There was a time when DEPC was added to soda pop as a source of effervescence. That use was banned in the USA in 1972.

[Priyanka]

I have no idea as to what should I do I have taken as many precautions as possible but my RNA looks like trash in the gel.. Trizol worked once but I dont know if that would happen another time... Please someone tell me what should I do to get good quality of RNA.. I did keep my DEPC at 37C O/N (like Sumit said) lets see if that was the problem...
I have deadlines to meet... and other experiments lined up but no RNA please someone help me...

[Priyanka]

DEPC problem solved .... Glassbottles should be used for preparing it!! and Sumit O/N on 37C worked well Thanx for the Tip
Cheers
Priyanka

[Jon Moulton]

Great, Priyanka -- on to the next experiment!

[Priyanka]

Hi John,..could you please send me a copy of the paper that you reffred (I dont have access to that i my libraray). I am sorry to bother you for this but this paper could help me understand more about things that I am dealing with
Cheers
Priyanka

[Jon Moulton]

Hi Priyanka,

I don't have a copy -- I pulled the citation from one of my old papers which I wrote shortly after reading the Henderson et al. paper. I'm afraid PubMed won't be much help, not even an abstract:
http://www.ncbi.nlm.nih.gov/pubmed/4350313

Perhaps there is a citation here which will be easier for you to access.

N J Leonard, J J McDonald, R E Henderson, M E Reichmann . Reaction of diethyl pyrocarbonate with nucleic acid components. Adenosine. Biochemistry. 1971 Aug 31;10 (18):3335-42

N J Leonard, J J McDonald, M E Reichmann. Reaction of diethyl pyrocarbonate with nucleic acid components. I. Adenine. Proc Natl Acad Sci U S A. 1970 Sep ;67 (1):93-8

A Vincze, R E Henderson, J J McDonald, N J Leonard . Reaction of diethyl pyrocarbonate with nucleic acid components. Bases and nucleosides derived from guanine, cytosine, and uracil. J Am Chem Soc. 1973 Apr 18;95 (8):2677-82

[Priyanka]

Thankx Jhon The publications are litlle old so they are not opening online i have forwarded my request to person incharged about the articles reffered...I`ll trouble you again if he is not able to get them ....
Cheers
Thankx again