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
What about EDTA? 0.1M?
What about EDTA? 0.1M?
1 mM