1. Purify plasmid from bacteria.
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.
2. Linearize 200 micrograms of plasmid DNA with the appropriate restriction enzyme digest.
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 degradation.