X-linker ratio in Acrylamide gels

Matt Thomas Thomas at molbio.uoregon.edu
Mon Mar 24 05:44:57 EST 1997

I'm currently having fun trying to look at misincorporations in RNA. 
One way we have thought of doing this is to use a mobility difference in
the two types of RNA.  IN fact we have observed a slight difference in a
few cases (about 1mm).  My question is can you increase the separation
of RNAs in an acrylamide gel my incresing the ammount of crosslinker?  I
do know of one paper from the Luse lab where they were looking at short
RNAs (<20 bases) and used a ratio of 25:3.  With these gels they could
observed a difference in mobility based on sequence.  

My RNAs are longer (about 45mers) so I tried their ratio but at a lower
percentage (10%).  What I got was a gel with the urea crashing out of
the gel and the gel being worthless.  Does anyone out there have any
experince with gels that have a higher ratio of acryl to bis?  How can
you keep the urea from crashing?  How high of ratios in denaturing gels
can you use?

Thanks for any comments

Matt Thomas

Thomas at molbio.uoregon.edu

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