Preparation of Bacterial Plasmid DNA

JoAnne Engebrecht1, J.S. Heilig2, Roger Brent3

1 State University of New York, Stony Brook, New York, 2 University of Colorado, Boulder, Colorado, 3 The Molecular Sciences Institute, Berkeley, California
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 10.3
DOI:  10.1002/0471142735.im1003s27
Online Posting Date:  May, 2001
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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.

     
 
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Table of Contents

  • Basic Protocol 1: Miniprep by Alkaline Lysis
  • Basic Protocol 2: Large‐Scale Crude Prep by Alkaline Lysis
  • Basic Protocol 3: Purification by CsCl/Ethidium Bromide Equilibrium Centrifugation
  • Support Protocol 1: Growing an Overnight Culture
  • Support Protocol 2: Growing Larger Cultures
  • Support Protocol 3: Monitoring Growth with a Spectrophotometer
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Miniprep by Alkaline Lysis

  Materials
  • Plasmid‐bearing E. coli strain
  • recipeLB medium (see recipe) containing ampicillin or other appropriate antibiotic
  • recipeGlucose/Tris/EDTA (GTE) solution (see recipe)
  • TE buffer ( appendix 2A)
  • NaOH/SDS solution: 0.2 N NaOH/1% SDS (prepare just before use)
  • recipePotassium acetate solution, pH 4.8 (see recipe)
  • 95% and 70% ethanol
  • TE buffer containing 0.1 mg/ml RNase or recipe10 mg/ml DNase‐free RNase (see recipe)
  • 1.5‐ml disposable microcentrifuge tubes

Basic Protocol 2: Large‐Scale Crude Prep by Alkaline Lysis

  Materials
  • Plasmid‐bearing E. coli strain
  • recipeLB medium (see recipe) containing ampicillin or other appropriate antibiotic
  • recipeGlucose/Tris/EDTA (GTE) solution (see recipe)
  • Hen egg white lysozyme
  • NaOH/SDS solution: 0.2 N NaOH/1% SDS (prepare just before use)
  • recipePotassium acetate solution, pH ∼5.5 (see recipe)
  • Isopropanol
  • 70% ethanol
  • Sorvall GSA, GS‐3, or Beckman JA‐10 rotor (or equivalent)
  • Sorvall SS‐34 or Beckman JA‐17 rotor (or equivalent)

Basic Protocol 3: Purification by CsCl/Ethidium Bromide Equilibrium Centrifugation

  Materials
  • DNA pellet from crude prep (see protocol 2)
  • TE buffer ( appendix 2A)
  • Cesium chloride
  • 10 mg/ml ethidium bromide
  • CsCl/TE solution: 100 ml TE buffer + 100 g CsCl
  • recipeDowex AG50W‐X8 cation exchange resin (see recipe)
  • 0.2 M NaCl prepared in TE buffer
  • 100% and 70% ethanol
  • Beckman VTi65 or VTi80 rotor (or equivalent)
  • 5‐ml quick‐seal ultracentrifuge tubes
  • 3‐ml syringes with 20‐G needles
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Figures

Videos

Literature Cited

Literature Cited
   Birnboim, H.C. 1983. A rapid alkaline extraction method for the isolation of plasmid DNA. Methods Enzymol. 100:243‐255.
   Birnboim, H.C. 1988. Citation classic. Current Contents (Life Sciences) 31(45):12.
   Birnboim, H.C. and Doly, J. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl. Acids Res. 7:1513‐1523.
   Heilig, J.S., Elbling, K.L., and Brent, R. 1998. Large‐scale preparation of plasmid DNA. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 1.7.1‐1.7.16. John Wiley & Sons, New York.
   Radloff, R., Bauer, W., and Vinograd, J. 1967. A dye‐buoyant‐density method for the detection and isolation of closed circular duplex DNA: The closed circular DNA in HeLa cells. Proc. Natl. Acad. Sci. U.S.A. 57:1514‐1521.
   Raleigh, E.A. 1989. Selected topics from classical bacterial genetics. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 1.4.1‐1.4.14. John Wiley & Sons, New York.
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