Plasmid Ligation Using a Sacrificial Insert

摘要

In the course of studying oxido-squalene cyclase enzymes, members of this laboratory have often ligated DNA cleaved with two restriction enzymes to minimize the occurrence of self-ligated plasmids. In cases where the two restriction sites are adjacentor overlapping in a multicloning site, it is often difficult to completely digest the DNA with the second enzyme. In plasmids lacking a reporter gene that allows visual screening for insertion events, this can lead to a laborious characterization by minipreps or hybridization.