Summary: Transgenic and Gene Targeting Core
9500 Gilman Drive, MC 0687
La Jolla, CA 920930687
Current as of Aug. 2008
Preparing Plasmid DNA for Electroporation
PLEASE DO NOT USE QUIAGEN COLUMNS FOR ANY PURIFICATION STEPS.
1. Isolate plasmid DNA by CsCl density gradient purification. (Maniatis, et al. Molecular
Cloning, pages 93 and 94) OR USE Promega Midi prep kit, called "Pure Yield Plasmid
Midi Prep." (Catalog #A2492)
2. Linearize 500 mg of purified plasmid DNA (the core needs 200 g DNA) with appropriate
restriction enzyme/s in a total volume of 500 ml (1 unit enzyme/ mg DNA). After
incubating for approximately 2 hr, analyze 1 ml of the reaction by agarosegel
electrophoresis to ensure the DNA is completely linearized.
Please present a photo of this gel to the Core.
3. Add equal amounts of phenol (phenol means phenol equilibrated with buffer and
containing 0.1% hydroxyquinoline and 0.2% beta mercaptoethanol) to the reaction