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-   -   Native gel or Agarose for RNA EMSA? (http://www.molecularstation.com/forum/electrophoretic-mobility-shift-assay-forum/84166-native-gel-agarose-rna-emsa.html)

Identity_crisis 11-30-2011 06:11 AM

Native gel or Agarose for RNA EMSA?
 
Hi there,

I am going to do a biotinylated RNA EMSA. I would like to know what is the difference and how does it matter if I go for a native gel or an agarose gel.

Also, do you guys make individual components of a buffer (EDTA, DTT, APS) in DEPC treated water?

Identity_crisis 11-30-2011 06:29 AM

Re: Native gel or Agarose for RNA EMSA?
 
What percentage gel shall I make? My RNA is 2.4kb and protein is 68kD

mmorgan 11-30-2011 04:24 PM

Re: Native gel or Agarose for RNA EMSA?
 
Quote:

Originally Posted by Identity_crisis (Post 437417)
Hi there,

I am going to do a biotinylated RNA EMSA. I would like to know what is the difference and how does it matter if I go for a native gel or an agarose gel.

Also, do you guys make individual components of a buffer (EDTA, DTT, APS) in DEPC treated water?


For EMSA you need to run either a native polyacrylamide or a native agarose gel. People often use 0.5x TBE as a buffer.

It's probably not absolutely necessary to use DEPC water, Milli-Q should be sufficiently pure (assuming your glassware isn't contaminated w/ RNaseA).

I think you will have trouble detecting a mobility shift for a 2.4 kb RNA on a polyacrylamide gel, so you may need to go for an agarose gel. The alternative is to use a fragment of the RNA that you think contains the binding site.


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