DNA in lysate - 293 lysis (Jul/18/2007 )
I am doing 293 cells lysis with Ripa buffer. My problem is DNA, I got so much.
even after sonication my lysate is still thick.
I spin it but it doesn't pellet.
increase the amount of RIPA buffer
you may try this one for total extraction of proteins :
NaCl 150mM (3ml NaCl 1M)
EDTA pH8 2mM (160µl EDTA 250mM)
NP40 1% (2ml NP40 10%)
Tris HCl 50 mm pH 7.5
ddH2O qsp 20ml
just before use add for 1ml of buffer :
-5µl DTT 1M
-1µl PMSF 100mM
use 300µl of lysis buffer for about 5 millions of cells
incubate cells + lysis buffer 10' on ice
Centrifuge 14000 rpm 30' 4°C
discard the pellet
the extract is ready for quantification and more analysis
i posted the protocol here