RNase without RNase

Jim Kami jakami at ucdavis.edu
Fri Feb 18 17:53:19 EST 2000


We have also been seeing band shifts and distortions. This apperently has been going on for
years and everyone just assumed it was due to carbohydrates in the sample. Recently I found
this paper which started us looking at our RNase treatments. We'd like to get rid of the RNA
to minimize it's effect on restriction digests.

Benore-Parsons, Marilee; Ayoub, Melissa A..  Presence of RNase A causes aberrant DNA band
shifts.       Biotechniques, v.23, n.1, 1997.:128-131.
    Abstract:    RNase A, which is routinely added during DNA purification to reduce
contaminating RNA, causes shifting of DNA bands in agarose gets. DNA band sizes on agarose
gels increase as much as 10%-20% when RNase A is present. The low concentrations of RNase A
typically used to purify DNA cause shifting of select DNA bands, in contrast to higher
concentrations of RNase A where all bands are shifted and smeared The binding of RNase A to
the DNA is specific and the degree of the shift varies; not all DNA bands are retarded, and
the deviation is more pronounced in certain buffers. Other proteins, such as bovine serum
albumin or proteinase K, do not induce DNA band shift, suggesting the interaction is
specific. The binding of RNase A to DNA is reversible. The formation of RNase:DNA complexes
may affect experiments involving DNA:protein interactions such as gel shift, footprinting and
filter binding assays. Researchers performing DNA characterization from miniprep protocols
should be aware that RNase may cause the apparent sizes of DNA fragments to be altered and
obscure the presence of very small cloned fragments.

Jim Kami

Sergio wrote:

> What kind of problem you've had?
> We have been using RNAase A for years without any problem, but recently the digestions
> with RNAase produce a DNA shift in the agarose gels. We've tried RNAase from Roche and
> Pharmacia without any difference. Has anybody had the same problem?. The only solution we
> found is to purify the DNA after digestion, using phenol.
> We prepare the RNAase by the Maniatis method: 10 mg/ml in 10 mM Tris pH 7.5 15 mM NaCl.
> Boiling 15 minutes and cooling down slowly.
> Any sugestion??
>
> Thanks in advance
> Sorry for my bad english
> Sergio
>
> Jim Kami wrote:
>
> > Hi,
> >     We've had a problem in our lab recently with our genomic DNA
> > isolations. Turns out, the cause is the RNase A we've been using.
> > Changing suppliers and prep. procedures hasn't helped.  I was wondering
> > if anyone knows of a way to eliminate RNA from genomic DNA preps without
> > using RNase ?
> >
> > Thanks in advance.
> >
> > Jim Kami
> > Dept. of Agronomy
> > University of California @ Davis
> > Davis, CA   95616





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