Ligation problems

Todd Miller todd33 at ix.netcom.com
Tue Jul 28 21:41:45 EST 1998


Sometimes genomic DNA can be a bear to clone.  I've overcome
similar problems by trying different bugs, but this is mainly
an empirical thing.  I also check the ligation reaction by
PCR, using primers complementary to the vector and flanking
the insert.  If you are having problems with too many re-
circularization events (ie, just the plasmid becoming a 
circle without an insert), you might try to design a strategy
that would allow you to restrict your finished ligation to
linearize these unwanted ligations (as long as the site is
destroyed with the desired ligation, and there are no sites
in the inserted DNA).

Good luck.  I would try different bugs first.

Todd Miller, PhD
University of Miami School of Medicine



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