**4.5.2 Plasmid based approach**

Cloning longer (100-500 bp) homology arms into the targeting construct is an alternative approach to generating a linear targeting vector. Using this type of construct is also useful for cloning sequence from BAC DNA into high copy plasmids for subsequent modification and use for in vitro cell targeting (i.e. embryonic stem cells). The linear targeting construct is released from the vector by restriction enzyme digest and gel purified. This approach reportedly reduces base changes that are sometimes generated during PCR.
