Answer to Question #118941 in Biochemistry for Wendy Dadson

Question

Why must the restriction enzyme used for the digestion of TOL plasmid be inactivated immediately after the restriction experiment? How could this be achieved?

Answer 1

Incomplete inactivation of the restriction enzyme can cause ligation to fail. Also if the insert carries the restriction site it may be damaged by restriction enzyme. The common way to inactivate the restriction enzyme is heat treatment (in accordance to inactivation protocol per the manufacturer), but if the enzyme is heat-resistant the vector should be purified by phenol-chloroform extraction or extracted from agarose gel after electrophoresis.