Endonuclease activity: Crude extraction from E.coli

Mikhail Alexeyev MAlexeyev at biost1.thi.tmc.edu
Wed Aug 2 09:53:53 EST 1995


Hi, Patrice:

In a past I found useful the following technique:
1. Spin down 1.5 ml of stationary-phase E. coli culture in Eppendorf tube
2. Resuspend pellet in 0.5 ml ice-cold 20 mM Tris pH 7.4-7.6, 50 mM NaCL,
5-10 mM 2-mercaptoethanol
3. sonicate on ice (time and intensity depends on culture and model of
sonicator, generally 6 x 10 sec with 10 sec. intervals, 50% pulse duration
and 50-90 power is OK in most cases)
4. spin down in microcentrifuge for 2 min @ max speed at 4 C (room t works
as well)
5. incubate 10 ul of lambda DNA (0.1 ug/ul in appropriate restriction
buffer) with 3 ul of resulting lysate for 10-30 min @ 37 C
6. run the gel

This procedure worked in my hands with about dosen of naturally occuring
producers of restriction enzymes as well as with genes cloned in E. coli.
In a latter case contaminating nucleases never presented a problem.
However, if this happens, some old remedy suggests to add t-RNA to titer
out nonspecific nucleases. 
Some adjustments might be necessary with above procedure (increase amount
of pellet, change buffer pH, salt, etc 

Hope, this will help.

M. Alexeyev


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

> I'm working on a restriction/methylation system in Rhizobium. A construct
> where the methylase has been knocked down and the endonuclease is intact,
> has been introduced into DH10B cells. The culture is growing slowly and
> many cell wall debris are present in the culture. This is in favour of an
> active endonuclease.
> 
> 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.
> 
> Thanks in advance for any help
> 
> Patrice Rochepeau, PhD            e-mail: prochepe at acs2.acs.ucalgary.ca
> University of Calgary
> 2500 University Dr.N.W.
> Calgary, AB, T2N 1N4
> Canada



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