Will DEPC interfere with PCR reaction

[jgleema]

Hi,
I isolated RNA, and in the steps involved used DEPC treated water. But while using that in RT-PCR, will DEPC interfere with the reaction? Is that necessary to aliquot the primers also in DEPC treated water?

[admin]

Hello Jgeleema,
have you inactivated DEPC/Rnases with autoclaving?

DEPC should be ok in your PCR after dilution, but MiliQ water or RNAse Free water should be better than DEPC treated water as DEPC only inactivates RNases without removing them.

Some inactivated Rnase can remain in solution and be activated after DEPC is damaged/removed.

Excess DEPC is destroyed by autoclaving to 121 C however it is hydrolyzed into pyrocarbonate and carbon dioxide. Ethanol is also generated as a side-product which although evaporates from solution may inhibit your PCR reaction a bit.

Some side products remain still after autoclaving and spent reagent contributes significantly to the water's total organic carbon (TOC) content.

[jgleema]

Hi admin,
Thank you for your reply. It was very useful to me.
Thank you.

[navidagar]

Hi !
i'm just strted working with rna wants to know about the depc treatment and if it is possible to work without it.

[Jon Moulton]

DEPC reacts with nucleic acid bases, alkylating them. It is best to autoclave water after DEPC treatment, as this will decompose the unreacted DEPC.

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