Does unautoclaved DEPC inhibit northern blotting? - (Aug/23/2010 )
I use DEPC treated H2O for all of the buffers from electrophoresis to immunodetection during northern blotting. If I stop autoclabing DEPC treated H2O or buffers made from it, will it have a negative impact on the blots? I know carboxymethylation of purines by DEPC inhibits translation but what about migration in the gel, RNA:RNA or DNA:RNA hybridization or immunodetection? It's not that I am lazy but we've all of the sudden have a very limited access to an autoclave.
DEPC will break down in the presence of water and heat, you can just heat the solution for a while (I don't know how long would be suitable). The major issue is of course that DEPC is quite toxic, so I am not sure that you would want it being realeased into the lab space every time you open the bottles for you to breathe in.
yekaterina poloz on Mon Aug 23 20:55:17 2010 said:
I use DEPC treated H2O for all of the buffers from electrophoresis to immunodetection during northern blotting. If I stop autoclabing DEPC treated H2O or buffers made from it, will it have a negative impact on the blots? I know carboxymethylation of purines by DEPC inhibits translation but what about migration in the gel, RNA:RNA or DNA:RNA hybridization or immunodetection? It's not that I am lazy but we've all of the sudden have a very limited access to an autoclave.
You need to autoclave it to inactivate the trace DEPC. DEPC will react with anything that has a -N, -O, or -S, so it may screw up your RNA bases. I would autoclave to be safe, especially if you're using it to store RNA in.
-b