Endonuclease activity: Crude extraction from E.coli
MAlexeyev at biost1.thi.tmc.edu
Wed Aug 2 09:53:53 EST 1995
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
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.
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
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