Preparation of Genomic DNA from Plant Tissue

Eric Richards1, Mark Reichardt2, Sharon Rogers2

1 (CsCl preparation) Washington University, St. Louis, Missouri, 2 (CTAB preparation) Lakeside Biotechnology, Chicago, Illinois
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 2.3
DOI:  10.1002/0471142727.mb0203s27
Online Posting Date:  May, 2001
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Abstract

This unit describes two methods for preparing genomic DNA from plant tissue. In the first method, plant cells are lysed with ionic detergent, treated with protease, and subsequently purified by cesium chloride (CsCl) density gradient centrifugation. The second method is based upon a series of treatments with the nonionic detergent cetyltrimethylammonium bromide (CTAB) to lyse cells and purify nucleic acid. Nucleic acid is recovered from the final CTAB solution by isopropanol or ethanol precipitation. The first method, although somewhat more lengthy, results in highly purified nucleic acid. The second method requires fewer manipulations, results in very high yields (˜10‐fold higher per gram fresh tissue depending on species and condition of starting material), and produces DNA that is less pure but nonetheless suitable in quality for use in many molecular biology manipulations.

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of Plant DNA Using CSCL Centrifugation
  • Alternate Protocol 1: Preparation of Plant DNA Using CTAB
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Preparation of Plant DNA Using CSCL Centrifugation

  Materials
    For recipes, see Reagents and Solutions in this unit (or cross‐referenced unit); for common stock solutions, see appendix 22; for suppliers, see appendix 44.
  • Plant tissue, fresh
  • Liquid nitrogen
  • recipeExtraction buffer (see recipe)
  • 10% (w/v) N‐lauroylsarcosine (Sarkosyl)
  • Isopropanol
  • TE buffer, pH 8.0
  • Cesium chloride
  • 10 mg/ml ethidium bromide
  • CsCl‐saturated isopropanol (equilibrate over a CsCl‐saturated aqueous phase)
  • Ethanol
  • 3 M sodium acetate, pH 5.2
  • 250‐ml centrifuge bottle
  • 55°C water bath
  • Beckman JA‐14, JA‐20 or JA‐21, and VTi80 rotors (or equivalents)
  • 5‐ml quick‐seal ultracentrifuge tubes
  • 15‐G needle and 1‐ml syringe

Alternate Protocol 1: Preparation of Plant DNA Using CTAB

  Additional Materials
    For recipes, see Reagents and Solutions in this unit (or cross‐referenced unit); for common stock solutions, see appendix 22; for suppliers, see appendix 44.
  • recipeCTAB extraction solution (see recipe)
  • CTAB/NaCl solution (unit 2.4)
  • recipeCTAB precipitation solution (see recipe)
  • 2% (v/v) 2‐mercaptoethanol (2‐ME)
  • recipeHigh‐salt TE buffer (see recipe)
  • 24:1 (v/v) chloroform/octanol or chloroform/isoamyl alcohol
  • 80% ethanol
  • Pulverizer/homogenizer: mortar and pestle, blender, Polytron (Brinkmann), or coffee grinder
  • Organic solvent–resistant test tube or beaker
  • 65°C water bath
  • Beckman JA‐20 rotor or equivalent or microcentrifuge
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Figures

Videos

Literature Cited

Literature Cited
   Dellaporta, S.L., Wood, J., and Hicks, J.B. 1983. A plant DNA minipreparation: Version II. Plant Mol. Biol. Rep. 1:19‐21.
   Fang, G., Hammar, S., and Grumet, R. 1992. A quick and inexpensive method for removing polysaccharides from plant genomic DNA. BioTechniques 13:52‐56.
   Jones, A.S. 1953. The isolation of bacterial nucleic acids using cetyltrimethlyammonium bromide (cetavlon). Biochim. Biophys. Acta. 10:607‐612.
   Murray, M.G. and Thompson, W.F. 1980. Rapid isolation of high‐molecular‐weight plant DNA. Nucl. Acids Res. 8:4321‐4325.
   Rogers, S.O. and Bendich, A.J. 1985. Extraction of DNA from milligram amounts of fresh, herbarium and mummified plant tissues. Plant Mol. Biol. 5:69‐76.
   Taylor, B. and Powell, A. 1982. Isolation of plant DNA and RNA. BRL Focus 4(3):4‐6.
   Watson, J.C. and Thompson, W.F. 1986. Purification and restriction endonuclease analysis of plant nuclear DNA. Methods Enzymol. 118:57‐75.
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