Extraction RNA (Tris-EDTA, DEPC-treated questions) - (Jun/03/2007 )

Hello, Everyone.

I am trying to extract DNA and RNA together from soil samples using Griffith (2000) method. The problem I have at the moment is not sure the concentration of Tris EDTA (pH 7.4) they used in the extraction. According to the paper, I found the company they bought the buffer and found out the concentration of Tris is 10mM and EDTA is 0.1M (which I thought too high!!!). Have any of you used this method to extract the RNA and DNA ? what kind of concentration do you use for extraction?

Another question is about DEPC-treated solution. I used to add 0.1% DEPC to the solution and leave it overnight in the fume cupboard, then autoclave twice to get rid of DEPC. But now, I notice that lots of people incubate the solution with 0.1%DEPC at 37oC and even shake overnight, then autoclave once. I am not sure once autoclave is it enough. Welcome any kind of discussion and idea. Thanks.

well i autoclave twice too. But 1 is fine for in vitro transcription assays.
Also EDTA can't vbe DEPC etreated as well as Tris as amines are destroyed by DEPC.
These solutions should be prepared with DEPC-treated water and clean material !.

i autoclave DEPC treated water 2 times at 121c, 1 atm for 3 hours

Thanks guys. Any idea about the Tris buffer concentration used for RNA extraction?

well normally Tris EDTA solutions are in 10mM tris