After plasmid preparation, the fragment to be subcloned is ligated into the
prepared vector. The ratio of prepared vector to prepared insert is estimated
by agarose gel electrophoresis
of 2 
l of each.
The ligation is performed using a T4 DNA ligase . The
enzyme, a single polypeptide, catalyzes the formation of a phosphodiester bond
between adjacent 3' hydroxyl and 5' phosphate termini in
DNA[112].
The appropriate amounts of vector and insert were incubated at 14psy176 C
overnight with 1 l ( 
20 Weiss units)
T4 ligase (Promega) in 1 
ligase buffer (50 mM Tris [pH
7.4], 10 mM MgCl 
, 20 mM DTT, 5 g/ml BSA, 1 mM ATP) in 20 l
volume.