Purification of Gene Targeting Vector DNA for Electroporation

1. Purify Plasmid DNA from Bacteria.
We recommend the either the Sigma-Aldrich GenElute HP Endotoxin Free  Plasmid Purification kit or the Qiagen EndoFree Plasmid Maxi kit for the purification of the targeting vector plasmid from bacteria. Please follow the directions in the kit. Electroporation of Qiagen purified DNA has been used successfully by a number of labs. Alternatively, plasmid DNA can be purified by CsCl banding.

2.  Linearize Circular Plasmid DNA

Linearize the circular plasmid targeting vector DNA with a restriction enzyme that cuts once in the plasmid vector backbone, not in the arms of homology that will mediate recombination. Digest a sufficient amount of plasmid DNA (~300 ug) to produce 200 ug for ES cell electroporation.
Run a DNA on a minigel to verify that digestion is complete.  Extract the DNA with 1 volume of phenol-chloroform, then with 1 volume of chloroform and precipitate by adding 0.1 volumes NaCl and 2 volumes cold ethanol. Resuspend the DNA in sterile TE (10 mM Tris-HCl, pH 8.0, 1.0 mM EDTA) at 1 mg/ml. Prior to electroporation, the Transgenic Core will verify the concentration and run it on a minigel to check the size.

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