Preparation of bacterial plasmid DNA.

J. Engebrecht, J. S. Heilig, R. Brent

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
JournalCurrent protocols in immunology / edited by John E. Coligan ... [et al.]
VolumeChapter 10
StatePublished - May 2001
Externally publishedYes

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Bacterial DNA
Plasmids
Ethidium
Agar Gel Electrophoresis
Ion Exchange
DNA
DNA Restriction Enzymes
Centrifugation
Bacteria
Growth
cesium chloride
caN protocol

ASJC Scopus subject areas

  • Medicine(all)

Cite this

Preparation of bacterial plasmid DNA. / Engebrecht, J.; Heilig, J. S.; Brent, R.

In: Current protocols in immunology / edited by John E. Coligan ... [et al.], Vol. Chapter 10, 05.2001.

Research output: Contribution to journalArticle

Engebrecht, J. ; Heilig, J. S. ; Brent, R. / Preparation of bacterial plasmid DNA. In: Current protocols in immunology / edited by John E. Coligan ... [et al.]. 2001 ; Vol. Chapter 10.
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