SDS Problem

[Peter Heifetz]

In article <youngl-0410941307080001 at 202.36.134.83>, youngl at hort.cri.nz
(Lester Young) wrote:

> Hi, we have a problem with our SDS coming out of solution whilst washing
> our membranes.  We hybridise in 5xSSC, 0.1%SDS, with Deinhardts, poly A
> and Herring sperm present, at 65C O/N.  We then wash twice with 2xSSC,
> 0.1%SDS at room temp.  This is where the problem arises.  The solution in
> the wash box goes cloudy, with flakes of what look like SDS, but the
> solution that's standing at room temp for the next wash, remains clear!
> 
> Any ideas what could be happening.  It may be a simple problem, but we're
> baffled and we think that it may be affecting the stringency of our
> washes.
> 

We've noticed something similar with protein samples in LDS. The detergent
comes out of solution at room temp when samples were prepared by acetone
precip in [brand new] plastic 13ml Sarstedt screw cap tubes, lyophilized,
and solubilized in 2% LDS / 40 mM Tris pH 7.4/ 5 mM EDTA.  The LDS still
precipitates even when the samples are transferred to 1.5 ml Eppys;
identical samples prepared wholly in Eppys do not show any precipitation. 
The LDS goes back into solution readily at 40 deg or so, and since the
samples are heated with B-mercapto right before loading it isn't really a
problem.  It is a bit unsettling to have one's "solubilized" samples
solidify, though...

I've checked with glass tubes and other plastic tubes and don't see this,
so it's something specific to the Sarstedt's.  Maybe they are made out of
the same stuff as your wash boxes.

-- 
Peter Heifetz
Duke University
Developmental, Cell, and Molecular Biology Group