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.
Transgenic
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