Appendix

APPENDIX 1J Preparation of Bacterial Plasmid DNA

  1. JoAnne Engebrecht (miniprep)1,
  2. J.S. Heilig (large-scale prep and CsCl/ethidium bromide)2,
  3. Roger Brent3

Published Online: 1 MAY 2001

DOI: 10.1002/0471142301.nsa01js10

Current Protocols in Neuroscience

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.

Author Information

  1. 1

    State University of New York, Stony Brook, New York

  2. 2

    University of Colorado, Boulder, Colorado

  3. 3

    The Molecular Sciences Institute, Berkeley, California

Publication History

  1. Published Online: 1 MAY 2001
  2. Published Print: FEB 2000

Abstract

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.