APPENDIX 1J Preparation of Bacterial Plasmid DNA
Published Online: 1 MAY 2001
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Neuroscience
How to Cite
Engebrecht, J., Heilig, J. and Brent, R. 2001. Preparation of Bacterial Plasmid DNA. Current Protocols in Neuroscience. 10:1J:A.1J.1–A.1J.10.
- Published Online: 1 MAY 2001
- Published Print: FEB 2000
The protocols in this unit describe methods for preparing bacterial plasmid DNA free from chromosomal DNA. The first is an alkaline lysis miniprep suitable for screening a moderate number of bacterial colonies by restriction endonuclease cleavage and agarose gel electrophoresis. The second is the first step to producing large amounts (milligrams) of plasmid DNA and is also based on alkaline lysis of the bacterial cells. The crude lysate generated in this protocol can be further purified by centrifugation using CsCl/ethidium bromide (CsCl/EtBr) equilibrium density gradients. Three support protocols provide information on how to grow overnight and larger cultures of bacteria, and how to monitor bacterial growth with a spectrophotometer. Other methods, some relying on commercially available ion-exchange columns, are discussed in the commentary.