methods for lysis of tissue culture cells

Dr. Kent Nastiuk nastiuk at rockvax.rockefeller.edu
Wed Jun 21 15:44:15 EST 1995


jlight at RESUNIX.RI.SICKKIDS.ON.CA (Dr. Jeff Lightfoot) wrote:
>Anybody have a method for lysing small quantities of lymphoblasts or
>fibroblasts for SDS-PAGE and Western blotting without boiling?  My main
>interest is a procedure that will completely shear DNA.  I've tried
>sonication, but our probe isn't that convenient to use because of the small
>volumes that I'm using (less than 100 ul).  Use of needle to shear the DNA
>works a bit, but there's still a lot of unsheared DNA present.  I'd like to
>use DNase, but the lysis buffer that I use has either SDS or urea or both,
>which I understand will denature DNase.  Thanks for your help.
>
>Jeff.
>
>"All too often a man's accumulated knowledge dies with him"
>
>        James A. Peters on the life and experiences of L. J. Stadler.
>
>
>

Have you tried using a sonicator bath?  this is what i routinely use for 
small volumes, though it will heat your sample and may take a while for 
whole cell lysates





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