How to make Tris RNAse Free? - (Jun/26/2006 )
Would you please tell me how I can make RNAse free Tris? I know I can't use DEPC for that.
if it is tris buffer , u may try autoclaving for one hour at 121 degree and the usual pressure in normal autoclaves
i am not sure of this but some people do it
hope u find it useful
The standard answer is to use a dedicated bottle of Tris base, and make the buffer up in DEPC treated water. You have to avoid introducing the RNAses, rather than removing them. Gloves. Clean glassware, spatulas, chemicals. Or you can buy RNAse free Tris solutions from, e.g., Ambion (now Invitrogen).
Thanks phage434 and phytoviridae!
"crazy but clean procedure"
Do it with 2 bottles : you have to wash with NaOH a dedicated bottle. Then wash it standard procedure. Then fill with water and treat with DEPC. Autoclave (removes tracesof detergents). Throw DEPC treated water away and refill with water 1 of the bottles in which you'll treat by DEPC (the other one will serve for preparation of tris).
autoclave both and ready for preparation
wash 2 bottles standard procedure. DEPC treat water in one of them and reserve the second to prepare Tris.
hi sayeh ! RNase is highly thermostable . it can only be destroyed by temperatures above 300 C so chemical treatment to remove RNAse is the best option. you can treat Triple Distilled Water with DEPC 2 hrs to over night. DEPC destroys the RNAse. autoclave it twice. autoclaving destroys DEPC. now you can use this water for preparation of DEPC. i agree with others that using RNAse free Tris is better and also keep reagetns bottles dedicated to RNA work.
RNA is very unstable in TRIS. that is why MOPS is used.
all the best.