blunt ligation problem

Robert M. Mihalek mihalek at formula1.smtp.anes.upmc.edu
Tue Aug 18 08:28:41 EST 1998


In article <1998Aug17.155745.6440 at leeds.ac.uk>
medbh at biovax.leeds.ac.uk writes:

> In answer to queries:  1.  To generate a blunt ended plasmid, I cut it with SmaI.  2.  I did also dephosphatase the plasmid.  This worked as I tested this by comparing normal digested plasmid to dephosphatased plasmid when transforming the bacteria.

So, you have a SmaI-(or some other blunt-end enzyme) cut plasmid with
and a SmaI-cut insert. The plasmid was dephosphorylated, yet you can't
get any subclones.

Maybe you did not completely inactivate (with heat) or remove (by
phenol extraction) the phosphatase? Can you subclone anything in this
plasmid, like some blunt-ended fragment that has worked in the past? In
other words, is there any positive control? 


Bob Mihalek
University of Pittsburgh





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