RNA formaldehyde agarose gel

redeamer aniko at ut.ee
Tue Jan 20 23:57:33 EST 2004


Good morning/evening/afternoon all!

I have one simple question, 

i am working with RNA-dependent RNA polymerase, and in its assay was
done as it should be, ive got a ladder of different RNA population,
however i have a feeling that my RNA is stucked up in the wells....
afterwards i realized that my loading buffer does NOT contain
formaldehyde that would react with -N= or :N- or any other possible
target for nucleophilic attack, so the formamide work (denaturing the
RNA at 65C 10 min) is totally useless!:((( And as a result RNA is
stucked up and i dont see a desired product (about 10KB)...
Am I right?

Another prob i have is that the resolution of my gel and its
discreteness is much more to be desired, i use phosphate buffer, 6%
formaldehyde, agarose system [BAD buffer goes hot and it stinks:)]...
However, i tried to use MOPS... but it does not contain
formaldehyde... got bad results, really bad..

I have heard about agarose-acrylamide gels, with a fantastic
resolution? Are there?.. and one professor at our fascility says that
it is a real art to make those gels, any comments on that?

Could anyone please suggest me a very NICE approach to solve those
problem?

Thank You in advance,

Drew



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