During ligation, DNA ligase will catalyze the formation of a phosphodiester bond between adjacent nucleotides only if one nucleotide contains a 5'-phosphate group and the other a 3'-hydroxyl group. Recircularization of plasmid DNA can therefore be minimized by removing the 5' phosphates from both ends of the linear DNA with calf intestinal phosphatase [110, 111]. As a result, neither strand of the duplex can form a phosphodiester bond . However, a foreign DNA segment with 5'-terminal phosphates can be ligated efficiently to the dephosphorylated plasmid DNA to give an open circular molecule containing two nicks. Because circular DNA (even nicked circular DNA) transforms much more efficiently than linear plasmid DNA, most of the transformants will contain recombinant plasmids.
For dephosphorylization, the vector digest (see section 3.3.1),
5 
l CIP buffer (1mM
ZnCl 
, 1mM MgCl , 10mM Tris, pH 8.3) and 1 l
calf intestinal phosphatase (Boehringer Mannheim) were added to an end
volume of 50 l and incubated at 37psy176 C for 30 minutes. An additional
batch of enzyme (1.5 l) was added and incubated for further 30 minutes.
The dephosphorylization was followed by a phenol extraction
(see section phenol) and an ethanol precipitation (see section
2.5.5).