Purification and Concentration of DNA from Aqueous Solutions

David Moore1

1 Baylor College of Medicine, Houston, Texas
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Appendix 1G
DOI:  10.1002/0471142301.nsa01gs06
Online Posting Date:  May, 2001
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Abstract

This unit presents basic procedures for manipulating solutions of single‐ or double‐stranded DNA through purification and concentration steps. These techniques are useful when proteins or solute molecules need to be removed from aqueous solutions, or when DNA solutions need to be concentrated for reasons of convenience. The , using phenol extraction and ethanol (or isopropanol) precipitation, is appropriate for the purification of DNA from small volumes (<0.4 ml) at concentrations lower than 1 mg/ml. Three support protocols outline methods to buffer the phenol used in extractions, concentrate DNA using butanol, and extract residual organic solvents with ether. An alternative to these methods is nucleic acid purification using glass beads and this is also presented. These protocols may also be used for purifying RNA. The final two alternate protocols provide modifications to the basic protocol that are used for concentrating RNA and extracting and precipitating DNA from larger volumes and from dilute solutions, and for removing low‐molecular‐weight oligonucleotides and triphosphates.

     
 
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Table of Contents

  • Basic Protocol 1: Phenol Extraction and Ethanol Precipitation of DNA
  • Alternate Protocol 1: Precipitation of DNA Using Isopropanol
  • Support Protocol 1: Concentration of DNA Using Butanol
  • Support Protocol 2: Removal of Residual Phenol, Chloroform, or Butanol by Ether Extraction
  • Alternate Protocol 2: Purification of DNA Using Glass Beads
  • Alternate Protocol 3: Purification and Concentration of RNA and Dilute Solutions of DNA
  • Alternate Protocol 4: Removal of Low‐Molecular‐Weight Oligonucleotides and Triphosphates by Ethanol Precipitation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Phenol Extraction and Ethanol Precipitation of DNA

  Materials
  • ≤1 mg/ml DNA to be purified
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol ( appendix 2A)
  • 3 M sodium acetate, pH 5.2 ( appendix 2A)
  • 100% ethanol, ice cold
  • 70% ethanol, room temperature
  • TE buffer, pH 8.0 ( appendix 2A)
  • Speedvac evaporator (Savant)

Alternate Protocol 1: Precipitation of DNA Using Isopropanol

  • sec‐butanol
  • Polypropylene tube

Support Protocol 1: Concentration of DNA Using Butanol

  Materials
  • Diethyl ether
  • DNA sample
  • TE buffer, pH 8.0 ( appendix 2A)
  • Polypropylene tube

Support Protocol 2: Removal of Residual Phenol, Chloroform, or Butanol by Ether Extraction

  • 6 M sodium iodide (NaI) solution (see recipe)
  • DNA in a 50‐ to 200‐µl volume
  • Glass bead suspension (see recipe)
  • Wash solution (see recipe)
  • TE buffer, pH 8.0 ( appendix 2A)
  • Incubator or water bath at 45°C
NOTE: The above materials are also available as commercial kits (e.g., Glas‐Pac, National Scientific Supply; GeneClean, Bio101; and Qiaex Gel Extraction Kit, Qiagen).

Alternate Protocol 2: Purification of DNA Using Glass Beads

  • 4 M ammonium acetate, pH 4.8
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Figures

Videos

Literature Cited

Literature Cited
   Eickbush, T.H. and Moudrianakis, E.N. 1978. The compaction of DNA helices into either continuous supercoils or folded‐fiber rods and toroids. Cell 13:295‐306.
   Hall, S.S. 1987. Going for the gene. The Boston Globe Magazine, Aug. 2, p. 20.
   Kirby, K.S. 1957. A new method for the isolation of deoxyribonucleic acids: Evidence on the nature of bonds between deoxyribonucleic acid and protein. Biochem. J. 66:495‐504.
   Marmur, J. 1961. A procedure for the isolation of deoxyribonucleic acid from microorganisms. J. Mol. Biol. 3:208‐218.
   Palmiter, R.D. 1974. Magnesium precipitation of ribonucleoprotein complexes. Expedient techniques for the isolation of undegraded polysomes and messenger ribonucleic acid. Biochemistry 13:3606‐3615.
   Penman, S. 1966. RNA metabolism in the HeLa cell nucleus. J. Mol. Biol. 17:117‐130.
   Vogelstein, B. and Gillespie, D. 1979. Preparative and analytical purification of DNA from agarose. Proc. Nat. Acad. Sci. U.S.A. 76:615‐619.
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