RNasing minilysates

K.C. Baker mbkxb at s-crim1.dl.ac.uk
Thu Jul 29 09:39:32 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

We got this with a batch of RNAase that was made up in the lab some months ago.
We cured the problem by adding the RNAase to the molten agarose when pouring 
the gel. Don't worry about the temperature, the enzyme renatures fine. Then just
restrict the miniprep (complete with RNA), and electrophorese as usual. The RNA
degrades on the way thru the gel. We use about 20ug of RNAase in a 50-100ml gel.
Cheers,
Ken.

-- 
Dr Ken Baker                              JANET : UK.AC.DL.SEQNET::MBKXB
Department of Protein Engineering       INTERNET : MBKXB at SEQNET.DL.AC.UK      
AFRC Institute of Food Research            TEL :        (+44) 734 357139
Reading Berks RG6 2EF                      FAX :        (+44) 734 267917



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