RNA Denaturation on PAGE

rogier stugerNOstSPAM at cellbiology.uni-frankfurt.de.invalid
Fri Feb 4 13:37:57 EST 2000


Hi Choona,
yep, you gotta keep it hot. 7M urea alone is not enough.
best thing is to prerun your gel while you're boiling your RNA,
and keep it hot.
Seuencing gel setups with metal plates are hot enough, but the
gels are horribly big and no fun to handle. I used minigel setups
(like in protein minigel systems) at 250 volts w/o additional
heating which worked OK. My bands got smeary when I ran below 200
V.
Running warm urea gels works fine for mRNA and rRNA, but tRNA
doesn't completely denature: a single tRNA will still give
multiple bands, the most prominent at about twice the size of the
tRNA itself.
Good luck,
Rogier Stuger
Mol Cell Physiol, Free Univ Amsterdam
rogier at biogate.com


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