RNase without RNase

Frank O. Fackelmayer Frank.Fackelmayer at uni-konstanz.de
Fri Feb 18 04:55:16 EST 2000


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??
>

Hi Sergio,
We´ve had that problem years ago, and did quite a lot to figure out what it was. Actually,
in our case it was just the amount of RNase we used. We prepared the RNase in exactely the
way you described, and used to use ul amounts of this RNase preparation for cutting RNA.
Reducing by a factor of 10 or more (up to 100 was not a problem) eliminated the problem
completely, and was still sufficient to get rid of the RNA.
Anyway, for convenience we have now switched completely to DNase-free RNase from Roche which
is supplied as 500ng/ul solution. This is, of course much more expensive than making up your
own from powder, but when you calculate the number of preps you can do with one tube (1ml
for 249 deutschmarks = approx. $120 is sufficient for at least 2000minipreps) the cost is
actually negligible.

Frank







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