Purification of Gene Targeting Vector DNA for Electroporation
We recommend 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.
1. Purify plasmid from bacteria.
2. Linearize 200 micrograms of plasmid DNA with the appropriate restriction
Run a DNA on a minigel to verify that digestion is complete. Extract
the DNA with phenol-chloroform, then with chloroform and precipitate by adding
NaCl and ethanol. Make sure you use fresh phenol with neutral pH for maximum
DNA recovery and highest cell viability in electroporation. Wash the DNA
pellet in 70% ethanol and allow to it air dry. Resuspend the DNA in sterile
TE (10 mM Tris-HCl, pH 8.0, 1.0 mM EDTA) at 2 mg/ml and deliver it to the
Transgenic Core for electroporation. Prior to electroporation, we will verify
the concentration and run it on a minigel to check the size and look for
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