| | |
Summary: UCSD Transgenic Mouse and
Gene Targeting Core
9500 Gilman Drive, MC 0687
La Jolla, CA 92093-0687
(858) 534-3178
Current as of 1/17/03
Preparing Plasmid DNA for Electroporation
1. Isolate plasmid DNA by CsCl density gradient purification. (Maniatis, et al.
Molecular Cloning, pages 93 and 94)
2. Linearize 500 µg of purified plasmid DNA (the core needs 200 g DNA) with
appropriate restriction enzyme/s in a total volume of 500 µl (1 unit enzyme/ µg DNA).
After incubating for approximately 2 hr, analyze 1 µl of the reaction by agarose-gel
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 tube.
4. Mix the contents of the tube until an emulsion forms (the organic and aqueous phases
may be mixed by vortexing when isolating small DNAs i.e. <10 kb, or by gentle shaking
when isolating DNAs of moderate size i.e. 10-30 kb).
5. Centrifuge the tube for 15 seconds in an eppendorf centrifuge at room temperature. If
|
