Minipreps of Plasmid DNA

JoAnne Engebrecht1, Roger Brent2, Mustak A. Kaderbhai3

1 State University of New York, Stony Brook, New York, 2 The Molecular Sciences Institutes, Berkeley, California, 3 University College of Wales, Penglais, Aberystwyth, United Kingdom
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 1.6
DOI:  10.1002/0471142727.mb0106s15
Online Posting Date:  May, 2001
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Abstract

Although there are a large number of protocols for the isolation of small quantities of plasmid DNA from bacterial cells (minipreps), this unit presents four procedures based on their speed and success: the alkaline lysis prep, a modification of the alkaline lysis prep that is performed in 1.5‐ml tubes or 96‐well microtiter dishes, the boiling method, and a lithium‐based procedure. A support protocol provides information on storing plasmid DNA.

     
 
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Table of Contents

  • Basic Protocol 1: Alkaline Lysis Miniprep
  • Alternate Protocol 1: Alkaline Lysis in 96‐Well Microtiter Dishes
  • Basic Protocol 2: Boiling Miniprep
  • Basic Protocol 3: Lithium Miniprep
  • Support Protocol 1: Storage of Plasmid DNA
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Alkaline Lysis Miniprep

  Materials
  • LB medium (unit 1.1) containing appropriate antibiotic (Table 97.80.4711)
  • recipeGlucose/Tris/EDTA (GTE) solution
  • TE buffer ( appendix 22)
  • recipeNaOH/SDS solution
  • recipePotassium acetate solution
  • 95% and 70% ethanol
  • 10 mg/ml DNase‐free RNase (optional; unit 3.13)
  • 1.5‐ml disposable microcentrifuge tubes

Alternate Protocol 1: Alkaline Lysis in 96‐Well Microtiter Dishes

  Additional Materials
  • TYGPN medium (unit 1.1)
  • 70% ethanol, ice‐cold
  • Isopropanol
  • 96‐well microtiter plates ( Dynatech PS plates or equivalent)
  • Multichannel pipetting device (8‐prong Costar; 12‐prong Titer Tek)
  • Multitube vortexer
  • Sorvall RT‐6000 low‐speed centrifuge, or equivalent, with microplate carrier in H‐1000B rotor

Basic Protocol 2: Boiling Miniprep

  Materials
  • recipeLB medium (unit 1.1) containing appropriate antibiotic (Table 97.80.4711)
  • recipeSTET solution
  • Hen egg white lysozyme
  • Isopropanol, ice‐cold
  • TE buffer ( appendix 22)
  • 10 mg/ml DNase‐free RNase (optional; unit 3.13)
  • 1.5‐ml disposable microcentrifuge tubes
  • Boiling water bath (100°C)

Basic Protocol 3: Lithium Miniprep

  Materials
  • recipeTELT solution
  • recipeLB medium (unit 1.1) containing appropriate antibiotic (Table 97.80.4711)
  • l:l (w/v) phenol/chloroform (unit 2.1)
  • 100% ethanol, prechilled to −20°C
  • TE buffer ( appendix 22)
  • 10 mg/ml DNase‐free RNase A (optional; unit 3.13)
  • 1.5‐ml disposable microcentrifuge tubes
NOTE: All steps are performed at room temperature.
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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. Meth. Enzymol. 100:243‐255.
   Birnboim, H.C. and Doly, J. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl. Acids Res. 7:1513‐1523.
   He, M., Kaderbhai, M.A., Adcock, I., and Austen, B.M. 1991. An improved and rapid procedure for isolating RNA‐free Escherichia coli plasmid DNA. Gene Anal. Tech. 8:107‐110.
   He, M., Wilde, A., and Kaderbhai. 1990. A simple single‐step procedure for small‐scale preparation of Escherichia coli plasmids. Nucl. Acids Res. 18:1660.
   Holmes, D.S. and Quigley, M. 1981. A rapid boiling method for the preparation of bacterial plasmids. Anal. Biochem. 114:193‐197.
   Ward, A.C. 1990. Single‐step purification of shuttle vectors from yeast for high frequency back‐transformation into E. coli. Nucl. Acids Res. 18:5319.
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