8. The targeting vector needs to be linearized for electroporation. The DNA is prepared by standard "maxiprep" procedures, for example, cesium chloride gradient centrifugation, or popular kits (such as Qiaex, Promega Wizard, Geneclean). Ethanol-precipitate the digested DNA before the electroporation, and dissolve the pellet in sterile TE. The concentration of the DNA should be around 1 pg/pL.
9. For the transient Cre electroporation, plasmid DNA can be used straight after "maxiprep" purification. In this case, the electroporation procedure is carried out according to the standard protocol provided. The Cre expressing vector can be coelectroporated along with a second selectable marker containing vector, or can be introduced alone (35). If a selection-based coelectroporating strategy for identifying cells taking up the Cre plasmid is used, then the cells should be plated at normal density (one cuvet into one or two 10-cm plates). Transient expression of the Cre recombinase in ES cell culture results in almost 100% excision between loxP sites placed a few kilobases apart (14).
10. When selecting for loss or gain of HPRT function, the cells should be maintained in the positive or negative selection prior to the selection switch that will assay the altered HPRT activity. The transiently expressed Cre-mediated excision is often mosaic. Therefore, a PCR screen should be carefully designed to detect such situations. Additionally, Southern blot analyses should be performed as a final check on candidate clones identified by PCR.
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