I prepare DEPC water in Milli Q (0.1%) following the standard protocol. After leaving the preparation overnight i autoclave the solution as it is required for DEPC breakdown. But what i have observed many times is that the pH of the autoclaved DEPC treated water is around 3.97-.4.08. I assumed initially that it may be due to ethanol or CO2 ( the products of DEPC breakdown). Hence i even tried keeping the bottles with lid loose in hood after autoclaving assuming that the CO2 gas will be diffusing out of the bottle and hence it would not get dissolved in water to make it acidic. But even after trying this the pH was acidic.
Hence can anyone please explain why is this happening because acidic water i believe will not be a good solvent for any molecular biology work.So i need to fix this so that i get the pH of DEPC water around neutral.
What is the pH of the water before preparation of DEPC.The pH of high purity water is very poorly controlled. Any trivial amount of any contaminant will shift it many pH units.
MilliQ water absorbs CO2 from the air. The value of CO2 in water at 25º C in equilibrium with air is 1.146 x 10-5 moles/liter (M). The carbon dioxide dissociates partially in water to produce equal concentrations of H+ and HCO3-: pH = 5.65.
Do not use pure DEPC-water directly for dissolving nucleic acids, use Tris/TE or PCR-grade water (e.g. from Sigma). It is OK for preparing buffers.
Thank you Jaroslaw for sharing the information.
The pH of the MilliQ before adding DEPC is 8.01. The pH of high purity water is poorly controlled because it lacks many ions? Is this the reason because of which the pH shifts drastically?
Also another open question is that: Many buffers change their pH after autoclaving but with time they get back to their actual pH values. So does the same apply for DEPC water as well?
Thank You Martin for your suggestion.
For isolation and storage of DNA/RNA i am using buffers already( QIAGEN) . But i prefer using DEPC treated water for preparations such as ( TAE/ ELECTROPHORESIS RUNNING BUFFER etc.)
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