Ligate, blunt ending and ligate

John Richard Seavitt jrseavit at artsci.wustl.edu
Wed Apr 1 14:48:15 EST 1998


On 1 Apr 1998, foc wrote:

> Wondered if i could first ligate the sticky end, then blunt the two others
> before a second ligation. In theory it sound ok? (or should I just blunt
> them both first and then ligate?)

I'd be nervous about this.  Ligase can catalyze the reverse reaction
(nicking) in the absense of dNTPs, so you'd have to purify the dna after
the first ligation.  Similarly, ligase is inhibited by salt, so you're
likely to have to desalt the DNA after blunting for the second ligation.
Probably need to purify a second time, since the polymerase is likely
exonuclease without dNTPs, and you don't want chew-back at the second
site.

Why bother?  Prep the vector by cutting with one enzyme, blunting (no need
for buffer change!), heat inactivate polymerase, and cut with second
enzyme (the one which will remain sticky).  Gel purify.  Same strategy for
insert... then ligate (easy since one end is sticky).

John Seavitt

"Seek enlightenment:  Thwack somebody over the head with a big stick."




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