Large‐Scale Preparation of Plasmid DNA

J.S. Heilig1, Karen L. Elbing2, Roger Brent3

1 University of Colorado, Boulder, Colorado, 2 Clark and Elbing LLP, Boston, Massachusetts, 3 The Molecular Sciences Institute, Berkeley, California
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 1.7
DOI:  10.1002/0471142727.mb0107s41
Online Posting Date:  May, 2001
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Abstract

Although the need for large quantities of plasmid DNA has diminished as techniques for manipulating small quantities of DNA have improved, occasionally large amounts of high‐quality plasmid DNA are desired. This unit describes the preparation of milligram quantities of highly purified plasmid DNA. The first part of the unit describes three methods for preparing crude lysates enriched in plasmid DNA from bacterial cells grown in liquid culture: alkaline lysis, boiling, and Triton lysis. The second part describes four methods for purifying plasmid DNA in such lysates away from contaminating RNA and protein: CsCl/ethidium bromide density gradient centrifugation, polyethylene glycol (PEG) precipitation, anion‐exchange chromatography, and size‐exclusion chromatography.

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of Crude Lysate by Alkaline Lysis
  • Alternate Protocol 1: Preparation of Crude Lysate by the Boiling Method
  • Alternate Protocol 2: Preparation of Crude Lysate by Triton Lysis
  • Basic Protocol 2: Purification of Plasmid DNA by CsCl/Ethidium Bromide Equilibrium Centrifugation
  • Alternate Protocol 3: Plasmid DNA Purification by PEG Precipitation
  • Alternate Protocol 4: Plasmid DNA Purification by Anion‐Exchange or Size‐Exclusion Chromatography
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of Crude Lysate by Alkaline Lysis

  Materials
  • LB medium or enriched medium (e.g., recipesuperbroth or recipeterrific broth; unit 1.1) containing ampicillin or other appropriate selective agent (Table 97.80.4711)
  • Plasmid‐bearing E. coli strain
  • recipeGlucose/Tris/EDTA solution (unit 1.6)
  • 25 mg/ml hen egg white lysozyme in recipeglucose/Tris/EDTA solution (prepare fresh)
  • 0.2 M NaOH/1% (w/v) SDS [prepare fresh from 10 M NaOH and 10% (w/v) SDS stocks]
  • recipe3 M potassium acetate solution, pH ∼5.5 (see recipe)
  • Isopropanol
  • 70% ethanol
  • Sorvall GSA, GS‐3, or Beckman JA‐10 rotor (or equivalent)
  • High‐speed centrifuge tubes with ≥20‐ml capacity (e.g., Oak Ridge centrifuge tubes)
  • Sorvall SS‐34 or Beckman JA‐17 rotor (or equivalent)

Alternate Protocol 1: Preparation of Crude Lysate by the Boiling Method

  • recipeSTET solution (unit 1.6)
  • 10 mg/ml hen egg white lysozyme in 25 mM Tris⋅Cl, pH 8.0 (prepare fresh)
  • Boiling and ice‐water baths
  • Sorvall HB‐4 rotor (or equivalent) and appropriate centrifuge tube

Alternate Protocol 2: Preparation of Crude Lysate by Triton Lysis

  • recipeSucrose/Tris/EDTA solution (see recipe)
  • 10 mg/ml hen egg white lysozyme in 25 mM Tris⋅Cl, pH 8.0 (prepare fresh)
  • 0.5 M EDTA ( appendix 22)
  • 10 mg/ml DNase‐free RNase (unit 3.13)
  • recipeTriton lysis solution (see recipe)
  • 1:1 (v/v) buffered phenol/chloroform (unit 2.1)
  • 24:1 (v/v) chloroform/isoamyl alcohol (unit 2.1)
  • Additional reagents and equipment for phenol/chloroform extraction (unit 2.1)

Basic Protocol 2: Purification of Plasmid DNA by CsCl/Ethidium Bromide Equilibrium Centrifugation

  Materials
  • Pellet from crude lysate of plasmid‐bearing bacterial cell culture (see protocol 1 or protocol 2 or protocol 32)
  • TE buffer, pH 7.5 ( appendix 22)
  • Cesium chloride
  • 10 mg/ml ethidium bromide ( appendix 22)
  • recipeCsCl/TE solution (see recipe)
  • recipeDowex AG50W‐X8 cation‐exchange resin (see recipe)
  • TE buffer (pH 7.5)/0.2 M NaCl
  • 100% and 70% ethanol
  • Beckman VTi65 or VTi80 rotor (or equivalent)
  • 5‐ml quick‐seal ultracentrifuge tubes
  • 3‐ml syringes with 20‐G needles
  • Additional reagents and equipment for ethanol precipitation (unit 2.1)
CAUTION: Ethidium bromide is a mutagen and environmental hazard. It should be handled carefully with gloves and disposed of properly. Methods for disposal vary between different institutions. Consult the institution's environmental safety office for the preferred means of storage and disposal of ethidium bromide–containing waste.

Alternate Protocol 3: Plasmid DNA Purification by PEG Precipitation

  • recipeGlucose/Tris/EDTA solution (unit 1.6)
  • 0.2 M NaOH/1% (w/v) SDS [prepare fresh from 10 M NaOH and 10% (w/v) SDS stocks]
  • 10 mg/ml DNase‐free RNase (unit 3.13)
  • recipe3 M potassium acetate solution, pH ∼5.5 (see recipe)
  • protocol 3Buffered phenol (unit 2.1)
  • protocol 324:1 (v/v) chloroform/isoamyl alcohol (unit 2.1)
  • 10 M ammonium acetate ( appendix 22)
  • recipePEG solution (see recipe)
  • 3 M sodium acetate, pH 5.5 ( appendix 22)
  • Sorvall SS‐34 or Beckman JA‐17 rotor (or equivalent)
  • Sorvall HB‐4 rotor
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Figures

Videos

Literature Cited

   Clewell, D.B. and Helinski, D.R. 1970. Properties of a deoxyribonucleic acid‐protein relaxation complex and strand specificity of the relaxation event. Biochemistry 9:4428‐4440.
   Clewell, D.B. and Helinski, D.R. 1972. Nature of ColE1 plasmid replication in Escherichia coli in the presence of chloramphenicol. J. Bacteriol. 110:667‐676.
   Tartoff, K.D. and Hobbs, C.A. 1987. Improved media for growing plasmid and cosmid clones. BRL FOCUS 9:2‐12.
Key References
   Birnboim, H.C. 1983. A rapid alkaline extraction method for the isolation of plasmid DNA. Methods Enzymol. 100:243‐255.
  These three references describe preparation of crude lysate by alkaline lysis.
   Birnboim, H.C. 1988. Citation classic. Current Contents (Life Sciences) 31(45):12.
  Describes preparation of crude lysate by boiling.
   Birnboim, H.C. and Doly, J. 1979. A rapid alkaline extraction method for screening recombinant plasmid DNA. Nucl. Acids Res. 7:1513‐1523.
  These two references describe preparation of crude lysate by Triton X‐100 lysis.
   Holmes, D.S. and Quigley, M. 1981. A rapid boiling method for the preparation of bacterial plasmids. Anal. Biochem. 114:193‐197.
  Describes plasmid DNA purification by CsCl/ethidium bromide centrifugation.
   Clewell and Helinski, 1970, 1972. See above.
  These two references describe plasmid DNA purification by PEG precipitation.
   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.
   Lis, J.T. 1980. Fractionation of DNA fragments by polyethylene glycol induced precipitation. Methods Enzymol. 65:347‐353.
   Lis, J.T. and Schleif, R. 1975. Size fractionation of double‐stranded DNA by precipitation with polyethylene glycol. Nucl. Acids Res. 2:383‐389. (Correction: the photograph of Fig. 1 should be interchanged with the photograph of Fig. 2. Lis and Shleif, 1975, Nucl. Acids Res. 2:757.)
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