Extraction and Precipitation of DNA


Publication Name:  Current Protocols in Human Genetics
Unit Number:  Appendix 3C
DOI:  10.1002/0471142905.hga03cs00
Online Posting Date:  May, 2001
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Abstract

This appendix presents protocols for phenol extraction and ethanol precipitation, methods that are routinely used to isolate DNA from biological sources or enzymatic reaction mixtures, to concentrate DNA samples, and to change from one solvent system to another. DNA isolation from simple aqueous solutions may be accomplished by ethanol precipitation alone.

     
 
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Table of Contents

  • Basic Protocol 1: Phenol Extraction
  • Basic Protocol 2: Ethanol Precipitation of DNA
  • Reagents and Solutions
  • Tables
     
 
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Materials

Basic Protocol 1: Phenol Extraction

  MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2; for suppliers, see suppliers appendix.
  • DNA sample to be extracted
  • 1:1 (v/v) phenol/chloroform or recipe25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol (see recipe)
  • 24:1 (v/v) chloroform/isoamyl alcohol
  • TE buffer, pH 8.0
CAUTION: Phenol and chloroform are hazardous; see appendix 2A for guidelines on handling, storage, and disposal.

Basic Protocol 2: Ethanol Precipitation of DNA

  MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2A; for suppliers, see suppliers appendix.
  • DNA sample to be precipitated
  • Appropriate monovalent cation solution (Table 3.0.1)
  • 100% and 70% ethanol, ice cold
  • TE buffer, pH 8.0
    Table 0.c.1   MaterialsMonovalent Cations Used for Precipitation of DNA

    Monovalent cation solution a Stock solution (M) Final conc. (M) Comments
    Ammonium acetate 10.0 2.0‐2.5 Use to prepare DNA for PCR; to precipitate oligonucleotides >50 bases; to reduce coprecipitation of dNTPs and small oligonucleotides. NH 4+ inhibits T4 polynucleotide kinase.
    Potassium acetate; pH 5.5  3.0 0.3 Use to isolate plasmids by isopropanol precipitation. Inhibits restriction enzymes.
    Sodium acetate; pH 5.2  3.0 0.3 Use to precipitate oligonucleotides <50 bases and to prepare DNA for most applications. Can interfere with PCR.
    Sodium chloride (NaCl)  5.0 0.2‐0.5 Use to precipitate samples containing SDS. Difficult to remove due to low solubility in 70% ethanol. Na+ inhibits T4 DNA ligase.

     aConsult appendix 2 for stock solution recipe.
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Literature Cited

Key References
   Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual. 2nd ed. pp. E.3‐E.15 Cold Spring Harbor Laboratory Press. Cold Spring Harbor, N.Y.
  Description of extraction and precipitation of nucleic acids.
   Wallace, D.M. 1987. Precipitation of nucleic acids. Methods Enzymol. 152:41‐48.
  Thorough description of precipitation techniques.
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