SDS extraction of whole cells

Michael L. Sullivan mlsulliv at facstaff.wisc.edu
Fri Jul 27 16:24:06 EST 2001


Warren,

I've found that I can shear the DNAsufficiently on fairly small SDS-PAGE
samples by using a 1 ml syringe to draw it up and push it back through an
18 or 22 guage needle.  I repeat this 20-30 times.  The sample can get
quite foamy, but that breaks down with a quick spin in the microfuge.  I've
done this on relatively small samples before-- on the order of 75-100 ul
with success.  Perhaps for a smaller sample this could be done with a
Hamilton syringe?

Hope this helps.

Mike

>Hi folks,
>	I am trying to do a Western blot on a set of cell lines and am trying
>to keep the protein concentration as high as possible.  The problem is
>that the DNA released on boiling in SDS makes the sample impossible to
>pipette accurately.
>	Aside from dilution, does anyone have a way to get the viscosity of
>this kind of extract down to a workable level?
>
>Thanks.
>
>Warren Gallin

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