How does one prepare nuclease free water?

Dear Rahul,

1. Get MilliQ (reverse osmosis purified) water. Double distilled water will do if you can't get MiiliQ water.

2. Add 1 ml DEPC (Diethylpyrocarbonate) per 1000 ml of MilliQ or double distilled water (i.e. to a final concentration 0.1%) and mix thoroughly.

3. Let the DEPC-mixed water incubate for 12 hours at 37°C.

4. Autoclave DEPC-mixed water for 15 minutes.

DEPC inactivates RNases by covalent modification. The autoclaving step inactivates the DNases and removes all traces of DEPC.

Hope this helps.

Regards,

Shaad

Also - autoclave the DEPC water two more times to be certain residual DEPC is eliminated (as a precautionary measure). Residual DEPC has been blamed for nucleic acid [RNA] carboxymethylation (see Integrated DNA Technologies literature), etc. So, if you do make your own DEPC-treated water, autoclave three times; or perhaps once for a longer period of time (e.g. 45 minutes). DEPC should ideally break fully down into water, ethanol and carbon dioxide if autoclaved long enough. DMPC is a safer alternative to DEPC - as DEPC is thought to be a carcinogen by some sources.

Thanks Shaad and Jack for your valuable suggestion.

thank you such  information

Thank you very much 

Thanks for the nice information; it would be more better if membrane filtration is done additionally - specifically if you use it for RT-PCR

Thanks 

May i know the storage temperature of DEPC treated water

I usually keep the DEPC-treated water at -20 C. I once used preserved DEPC water kept stored at - 20 C for 5 years and it functioned nicely

Thank you Shaad M Ahmad , I am planning to perform qPCR next days and this protocol will help.... Hopefully!

Thank you very much for the information.

1) In principle, milliQ water and double distilled water is essentially nuclease free if these systems are well maintained.

2) Often people do DEPC treatments just to be completely sure - since proving that water is completely nuclease free is ether difficult or takes more labour that the DEPC treatment.

3) If you store water in glass bottles, even if it has some traces of nucleases from occasional microbe material falling out from the air, such low amounts of protein will likely get completely inactivated in a few days due to adsorption to glass walls.

We are in same page micheal..i am thinking to autoclave the milliQ water and double distilled water one or two times to treasure no more rnase

Thank you so much for the above information.