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two-hybrid problems

Eugen C. Buehler buehler at pop.pitt.edu
Mon Oct 24 09:59:55 EST 1994


    I have recently been trying to use the two-hybrid yeast system (pEG202
and pJG4-5) to determine if two specific proteins interact.  Unfortunately,
the results I've gotten are somewhat erratic.  What I need to know is:

    1. Is the Gal promoter on pJG4-5 "leaky"?  Although I should only get
growth on the Gal leu- plates, I also get some growth on my Glu plates. 
(Note that I have checked to make sure growth does not take place with just
the pEG202 plasmid on leu- plates, gal/raf or glucose, so this is not the
problem.)

    2.  What is the optimum concentration of galactose/raffinose for drop
out plates?  I've been using 2% galactose and 1% raffinose, as recommended
in one protocol, but this concentration appears to be inhibiting growth on
my X-GAL plates.

  Many thanks in advance to anyone who can help me with these irksome details.

                                            Eugen Buehler
                                            Dept. of Biological Sciences
                                            University of Pittsburgh
                                            buehler at pop.pitt.edu



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