Endonuclease activity: Crude extraction from E.coli

Duncan Clark Duncan at genesys.demon.co.uk
Wed Aug 2 10:12:39 EST 1995


In article: <woodj-0108951134070001 at chevron23.bio.ucalgary.ca>  woodj at acs.ucalgary.ca (Julian Wood) writes:

> I'm looking for a quick method to make a crude extraction containing the
> endonuclease (and possibly get rid of non-specific endonuclease activity).
> 
> The extract will be used to test for the activity of the endonuclease on a
> target DNA.

Simple, harvest cells and resuspend in say 10mM KPi (pH 7.4), 10mM B-EtSH, 
0.1mM EDTA,10% glycerol. Sonicate, centrifuge and assay supernatant (possibly
using dilutions) against your DNA substrate. It ios not possible to get rid
of non-specific endonuclease unless you have a thermophilic enzyme ('cos then
you can heat treat the lysate). If DH10 is endA then the E.coli is as 
non-specific nuclease minus as you will get. If you need better then you will
have to purify your enzyme.

Duncan 
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