Re Transformation (Low efficiciency)

Louis Geller bbbbd at sprynet.com
Thu Aug 1 01:20:13 EST 1996


This is in response to your article in bionet.molbio.yeast.
        I am working on a similar mutant that is ts
(temperature-sensitive), and must also have its selection plasmid.
        At first, I had a low transformation efficiency. Since we've scaled
up the reactions, the efficiency is high. You may want to try this, and see
what happens.
        You may also try beta mercapto ethanol (5-20mM), or DMSO (5-10%) of
the final volume. I got these suggestions from Dr. Gietz' TRAFO page on the
web.
here is the URL:
http://www.umanitoba.ca/faculties/medicine/human_genetics/gietz/Trafo
.html
He suggests a 20 minute heat shock at 42 degrees, which can lessen the
required heat shock.

        Try varying the amount of carrier DNA. We also subject the cells to
continuous mixing (75rpm) during incubations with DNA and DNA/PEG. 
Also,
gentle vortexing at each addition.
        If I think of naything else, I'll email you.
        Louis Geller...
        Graduate Student, Cell & Molecular Biology
        CSU Long Beach

        lgeller at csulb.edu
        bbbbd at sprynet.com



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