Max,
16C as a temperature for ligation represents a compromise of optimal
temperature for annealing of short single stranded overhangs and that
for ligase activity. Blunt end ligations are traditionaly done at
room temperature as the first consideration is not important.
There does not appear to be a "routine ligation" as different combinations
of DNA fragments ligate with a widely different efficiencies. In approaching
a new combination of DNA fragments, I find it necessary to strive for the
highest possible ligation efficiency as difficult constructs are
as common as theoretically "simple" ones. Several-fold differences
in ligation efficiency can often make or break a plasmid construct
in my experience.
Jim
J. Graham
Biology and Chemistry Departments
Indiana Univeristy Bloomington
