E. coli cell free lysate
J. Arturo García-Horsman
arturo.garcia-horsman at uku.fi
Wed Apr 25 01:51:26 EST 2001
Wolfgang Schechinger wrote:
> Hi all,
> I'd like to prepare a cell free lysate from E. coli for a
> qualitative in vitro DNA replication experiment. Is there some
> easy procedure to try out (preferably lysing with triton
> and some chelators like EDTA or something alike)?
Here is how I have lysed E.coli. Hope that it would help. I send two
protocols, depending on your convenience. They are relatively old
procedures and some steps may be omited/changed, but it works like that.
Wash a cell pellet from a 250-500 ml cell culture (mid-log phase) with
0.15 M NaCl, 0.1 M EDTA (pH 8), and resuspend in 5 ml same solution. Add
lyzozyme to approx. 50 mg/ml and shake for about 20-40 minutes. (Here
you have to check if the lysozyme has completed its job taking at
various times, 50 to 100 ul to a glass tube and diluting 10-20 times
with water, the solution should not be turbid. You can compare with an
untreated cell suspension of the same density) When the lyzozyme has
worked, dilute to 25-50 ml with 10 mM KCl, 5 mM EDTA. Centrifuge at 5000
rpm for 15 min to eliminate unbroken cells, the supernatant is your
lysate. You can then add then the buffer and aditives of your choice.
Alternativelly, harvest, wash, resuspend and treat with lysozyme as as
above, instead of diluting, shell-freeze (dry ice-ethanol). Add 50 ml of
prewarmed (50 C) lysis buffer (1%SDS or 0.5% Triton X-100, 0.1 M
Tris-HCl, 0.1 M NaCl, pH 8), incubate 10 min at 50 degrees with constant
swirling. Centrifuge to eliminate devris.
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