S1/Mung Bean

Dr. Peter Gegenheimer PGegen at RNAworld.edu
Fri Mar 13 23:03:36 EST 1998


On Thu, 12 Mar 1998 23:01:00, wtd3 at columbia.edu wrote:

> Does anyone have suggestions for keeping S! or Mung Bean Nuclease single
> strand specific?  I am trying to chew back the overhang left from an
> NCO1 digestion, because I need to remove the ATG as part of a targeting
> construct.  However, with both enzymes I get a ton of degradation of my
> DNA, even at lower than recommended temp and enzyme concentrations.
> Alternatively, I'd be open to any other suggestions on how to
> specifically remove (NOT fill in) 5' overhangs generated by restriction
> enzymes.
>
> Thanks,
>
> Bill

First, since these enzymes digest only SINGLE-stranded DNA, you need
to make sure that the DNA is single-stranded; i.e. 50 mM salt, 5-10
mM Mg, pH 7 - 8.
Second, since both S1 and mung bean nucleases are rot-gut enzymes
which are maxinally active at pH 5+/-, you need to discard them and
use P1 nuclease. P1 is a member of the S1 family of
Zn-metallonucleases, but it is active at neutral pH, in the absence
of added Zn, and in a broad variety of buffers.

Since several people asked me for protocols, and I use P1 for
totally different purposes, I have found a number of references and
protocols which I will post next week. However, take a look at
Bramhill & Kornberg, Cell 52, 743-755 (1988); Kowalsi, NAR 12,
7071-7086 (1984); protocols in (Tal et al?) JMB 243, 179-189 (1994)
and (Rao et al?) Biochimie 73, 363-370 (1991) [you can omit their Zn
& EDTA].

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