Strange Bgal results

Kevin A. Morano kmorano at umich.edu
Thu Nov 7 14:49:45 EST 1996


Hi all,

   I have recently come upon a strange situation regarding a lacZ reporter
that I can't seem to figure out.  I have a well-characterized lacZ reporter
construct which has been used successfully in the lab for filter lift
assays.  I am employing it to screen a high-copy library for activators and
isolated 4 colonies which were blue on XGal plates.  My problem is that
after a few rounds of purification, only one of the four shows any activity
on XGal plates, and that one is quite robust.  In contrast, if I do a
filter assay, that same guy is near background levels and another one gives
a very strong signal.  In short, these two isolates display BGal activity
but only using different detection methods and fail to work on the
reciprocal method.  Furthermore, liquid Bgal assays essentially mirror the
filter-lift results.

   What gives?  I could understand if maybe one was stronger than the
other, but why would I get different results? The only difference I can see
between the two methods are that the XGal plates are pH 7.0 vs. the normal
SC pH of 5-6.  I would like to pursue these inserts but I would also like
to be able to use the same assay to test them.  I've checked the archives,
and there really isn't anything directly applicable, except for nearly
unanimous votes for the filter lift method over XGal plates.  As always, I
appreciate any advice I can get.

Thanks!
Kevin



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