E.coli cell breaking

Anne Spang spang at vms.biochem.mpg.de
Mon Jan 2 09:02:44 EST 1995


HI Marcel!
There are some other methods around.
You can use sonification! (which works pretty good, but you need a sonifier)
Another method would be freezing and thawing cycles . The result of these
technique may be better, if you do a lysozyme digest first. Resuspend your
E.coli pellet in 50 mM Tris-Cl, 0.5 mM EDTA, pH 8.0. Add per 0.1 ml:
0.0012 ml Lysozyme (10 mg/ml) 0.0005 ml PMSF 100 mM 0.01 ml Benzamidin 100
mM. Incubate for 5 min at RT. Freeze the cell suspension three times for
15 min at -20C with thawing cycles for 10 min at RT. Spin 5 min in a
benchtop centrifuge at 4C and the supernatants may contain your protein.
If you just want to analyse the cells on a gel, resuspend the cells in 2x
Laemmli sample buffer and incubate for 5 min at 95C.
If you run in trouble with to high DNA concentrations, you can DNAse in
the incubation buffer for the lysozyme digest!

Hope this helps!
Anne



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