SmaI/XmaI digests

Dr. Pamela Norton P_Norton at CALVIN.JCI.TJU.EDU
Mon Nov 14 17:20:45 EST 1994


Bionetters,

        As the subject line says, we have been having terrible problems
getting  a SmaI fragment re-subcloned; essentially we are trying to turn a
Sma fragment around. First attempt: cut plasmid with Sma, isolate both
bands, ligate back together, look for inserts in the right orientation.
Only one or two - but these don't cut with Sma, and sequencing says that a
single base is missing at both sites. Start again, with XmaI this time. Got
about four in the right orientation, these now cut only once with Sma or
Xma. Sequencing reveals that one of the sites (the same in all) has a base
missing. The cursed site is within a very GC-rich region, perhaps this is
the problem?
 
        I am literally tearing my hair out over this one. Does anyone know
whether Sma tends to nibble 5' or 3' ends? In other words, would it help if
we try to refill any nibbled ends before ligation? I'm thinking of
mutagenizing the site back into the plasmid with a single site present
(both are in coding sequence, so deletions are a major problem). Before I
resort to that, I thought that I would draw on the accumulated net wisdom.
All suggestions and relevant anecdotes are welcome. 

        Thanks,

                Pam Norton
------------------------------------------------------------------------------
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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