RNasing minilysates

nishir at ohsu.edu nishir at ohsu.edu
Thu Jul 29 14:40:20 EST 1993


In article <236j9tINN4ir at no-names.nerdc.ufl.edu> beatriz at pine.circa.ufl.edu
writes:
>Dear netters,
>
>When I do my minilysates I usually digest with about 2 ul of 20 mg/ml of RNase
H after the enzyme restriction digestion. The problem I've had is that if I'm
not careful and pipet more than 4 ul or so, my samples don't even get into 
>the gel (0.8% agarose-TAE). I get these huge aggregates that stay in the
wells. It is so annoying lately I just run my gel and then RNase the whole gel,
but this takes much longer and the bands from the inserts are usually somewhat
diffuse. Has anyone seen this before?  Does anyone know why it happens?
>
>Thanks in advance,
>
>Beatriz.
>beatriz at animal.ufl.edu
>beatriz at pine.circa.ufl.edu
>
Excuse me, but you should use RNase A instead of H.  RNase H will degrade RNA
in RNA/DNA duplexes, but will not digest single or double stranded RNA, which
is going to be the major contaminant in a mini-prep.

Rae



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