Sma/Xma problem-likely answer

Dr. Pamela Norton P_Norton at CALVIN.JCI.TJU.EDU
Fri Dec 16 16:46:08 EST 1994


        A month or so ago, I asked for suggestions regarding Sma or Xma
sites being damaged during what should have been a simple subcloning. Many
thanks to all who offered advice, anecdotes or just plain sympathy.
However, the apparent answer was offered by Paul Riggs of New England
Biolabs. He pointed out that the single base deletions we were seeing
sounded as if there was a selection for frameshifting. (Sure enough, the
insert in the orientation that we wanted would create a large open reading
frame with a signal sequence downstream of the the lacZ promoter in
bluescript. The Sma/Xma site that kept getting trashed is in the middle of
the signal sequence.) So, at his suggestion, we added glucose to more
tightly repress the lac promoter, and grew cells at 30-32oC to lower copy
number. Voila! Correct orientation with both Sma sites intact. Now we just
need to grow enough of this thing to take it to the final stage of
construction.

        Thanks again to all, especially to Paul. Another "Methods" success
story! Oh, and seasons greetings!

                Pam
------------------------------------------------------------------------------
Pamela A. Norton, Ph.D.             p_norton at lac.jci.tju.edu            
Assistant Professor of Medicine             
Thomas Jefferson University
1020 Locust Street, JAH 365
Philadelphia, PA  19107





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