Purification and Concentration of DNA from Aqueous Solutions

David Moore1

1 Massachusetts General Hospital, Boston, Massachusetts
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 10.1
DOI:  10.1002/0471142735.im1001s8
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. The , using phenol extraction and ethanol precipitation, is appropriate for the purification of DNA from small volumes (<0.4 ml) at concentrations <1 mg/ml. Isopropanol may also be used to precipitate DNA, as described in an alternate protocol. 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 is described here. These protocol may also be used for purifying RNA. The final protocols provide modifications to the that are used for concentrating RNA and extracting and precipitating DNA from larger volumes and from dilute solutions, and for removing ow‐molecular‐weight oligonucleotides and triphosphates.

     
 
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Table of Contents

  • Isolation and Purification of DNA
  • Basic Protocol 1: Phenol Extraction and Ethanol Precipitation of DNA
  • Alternate Protocol 1: Precipitation of DNA Using Isopropanol
  • Support Protocol 1: Buffering Phenol
  • Support Protocol 2: Concentration of DNA Using Butanol
  • Support Protocol 3: 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
  • DNA to be purified (≤1 mg/ml) in 0.1 to 0.4 ml volume
  • 25:24:1 phenol/chloroform/isoamyl alcohol (made with buffered phenol; first protocol 3support protocol)
  • 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

  Materials
  • 8‐hydroxyquinoline
  • Liquefied phenol
  • 50 mM Tris base (unadjusted pH ∼10.5)
  • 50 mM Tris⋅Cl, pH 8.0
  • Chloroform
  • Isoamyl alcohol

Support Protocol 1: Buffering Phenol

  Additional Materials
  • sec‐butanol
  • 25:24:1 phenol/chloroform/isoamyl alcohol (made with buffered phenol; first protocol)
  • Polypropylene tube

Support Protocol 2: Concentration of DNA Using Butanol

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

Support Protocol 3: Removal of Residual Phenol, Chloroform, or Butanol by Ether Extraction

  Additional Materials
  • recipe6 M sodium iodide (NaI) solution
  • DNA in a 50‐ to 200‐µl volume
  • recipeWash solution
  • TE buffer, pH 8.0 ( appendix 2A)
  • recipeGlass beads suspension
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

  Additional Materials
  • 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.
   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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