nucleus removal in Sf9 cells.

Karl Fischer tyr-2 at bones.biochem.ualberta.ca
Thu Dec 18 23:04:37 EST 1997


In article <34995b2f.260909792 at news.cc.uic.edu>, levenson at uic.edu (Victor
Levenson) wrote:

<snip>

"NP-40 will destroy the membrane"....hmmm. 

We usually lyse avian and mammalian cells in culture with 0.25% NP-40/50
mM NaCl/10 mM Tris-HCl (pH 7.5-8)/1 mM EDTA/ 8% sucrose and clarify in a
microfuge for 5 minutes in order to enrich for cytoplasmic virus particles
(nuclei will pellet). We do the lysis on monolayer cultures at 1 ml lysis
solution per plate (plate size 60-100 mm diameter) We do not vortex the
lysate.

Caveat - even the most gently lysis of cells can result in the release of
some nuclear and mitochondrial DNAs. If you wish to get rid of these and
DNAse will not affect your protein or purification procedure you can add
MgCl2 to your clarified lysate to 7 mM and then add DNAse I to 100 ug/ml
(final concentration) and incubate for 30-60 minutes at 37C. You may need
to clarify the lysate by a brief centrifugation after this incubation
period.

Or...you can do a hypotonic lysis as suggested by Dr. Levenson and just
spin out your nuclei in a microfuge (10 minutes, 4C).

Just some thoughts.
Good luck and happy holidays.

Karl the hepB guy



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