Stephen C. Dahl
stebby at welchlink.welch.jhu.edu
Wed Apr 15 13:00:34 EST 1998
René Bartz (bartr000 at mail.uni-mainz.de) wrote:
: Since severals weeks, we have a problem with our SDS-sample buffer. Our
: 1x sample buffer contains:
: 62.5 mM 0.5 M Tris Cl pH 6,8
: 10 % Glycerol
: 2% SDS
: 0.01% bromphenol blue
: The problem now is the development of flakes on the surface of the
: Perhaps is there an alternative protocol, which we can use.
I'll give you three :o)
As a grad student, Len Pagliaro, another grad student at the time, gave me
the following protocol. I'm not sure who to credit for this...
1X sample buffer
80 mM Tris pH 6.8
.002% bromphenol blue
add 5% BME/DTT just prior to use
As a postdoc in Daniel Branton's lab we used the following protocol..
5X sample buffer
250 mM Tris pH 6.8--------------------12.5 ml 0.5M stock
50% glycerol (well, almost 50%)-------bring total volume to 25ml
.02% bromphenol blue------------------5mg
add 10% BME prior to use
A friend of mine who worked with Harvey Lodish used yet a different
5X sample buffer
300mM Tris 6.8
I never asked how much BME he used
I, myself, have stuck with the Branton buffer above. I store 500 ul
aliquots in the -20 and add 50 ul BME after thawing. Note, however, that
this stuff is darn thick prior to the addition of the BME so I heat it at
65' C. to thaw.
As for your flakes, I can't say I've ever had that trouble. I have been
in labs where it is so darn cold that the 10 and 20% SDS stocks come out
of solution. These flakes tend to have neutral buoyancy though and aren't
restricted to the surface. Maybe your SDS can't swim.
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