S L Forsburg
forsburg at SALK.EDU
Thu Nov 7 11:58:00 EST 1996
Thomas O Westerling wrote:
> Hi All of you yeast lovers/haters (personally Im becoming a hater
> myself). I have a serious problem in making good lysates of S.pombe with
> the usual glassbead-disruption.
> If anyone could give me a hint on what to troubbleshoot I would be
> eternally (almost...:) grateful.
(1) You may not be using enough beads for the amount of buffer.
You want to have a slurry with no liquid on top of the beads--
sounds like you have too much buffer (100ul is more than enough.
50 is even better for the size cultures you are doing)
(2) are you breaking cells in microcentrifuge tubes? You will
get much better breakage if you move them into round bottom
15ml tubes. That's because you get better motion of the beads
to shear the cells than in the confined space of a microtube.
Feel free to email if you ahve more questions. You might like
to check out the references on our pombe methods web page (URL below.)
Susan L Forsburg PhD
MBVL, The Salk Institute
forsburg at salk.edu
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