**4.7.1 Identifying false positive recombinants**

In most cases, the template DNA used in the PCR reaction generating the linear targeting vector is eliminated by a DpnI digest, followed by gel purification. However, despite using DpnI to digest methylated DNA used as template in the generation of linear targeting vector by PCR, some plasmid template may be introduced into bacteria during the electroporation (Fig. 2). Identification of this contamination can be facilitated by gel electrophoresis of uncut BAC DNA isolated from kanamycin resistant clones. Even though the linear targeting construct is gel purified, the plasmid template can transform kanamycin resistant clones. For example, the undigested supercoiled plasmid DNA and the linear targeting vector can run at approximately the same molecular weight, thus contaminating the linear targeting vector during gel purification, as occurred in our studies.
