Purification and Concentration of DNA from Aqueous Solutions

David Moore1, Dennis Dowhan1

1 Baylor College of Medicine, Houston, Texas
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 2.1A
DOI:  10.1002/0471142727.mb0201as59
Online Posting Date:  August, 2002
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

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. The , using phenol extraction and ethanol (or isopropanol) precipitation, is appropriate for 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 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.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Section I: Manipulation Of DNA
  • Basic Protocol 1: Phenol Extraction and Ethanol Precipitation of DNA
  • Alternate Protocol 1: Precipitation of DNA Using Isopropanol
  • Support Protocol 1: Preparation of Buffered Phenol and Phenol/Chloroform/Isoamyl Alcohol
  • Support Protocol 2: Concentration of DNA Using Butanol
  • Support Protocol 3: Removal of Residual Phenol, Chloroform, or Butanol by Ether Extraction
  • Alternate Protocol 2: DNA Purification Using Silica Membrane Spin Columns
  • 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
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

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 (made with buffered phenol; see protocol 3)
  • 3 M sodium acetate, pH 5.2 ( appendix 22)
  • 100% ethanol, ice cold
  • 70% ethanol, room temperature
  • TE buffer, pH 8.0 ( appendix 22)
  • 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 ( appendix 22)
  • Chloroform
  • Isoamyl alcohol

Support Protocol 1: Preparation of Buffered Phenol and Phenol/Chloroform/Isoamyl Alcohol

  • sec‐Butanol
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol (made with buffered phenol; see protocol 3)
  • Polypropylene tube

Support Protocol 2: Concentration of DNA Using Butanol

  Materials
  • Diethyl ether
  • TE buffer, pH 8.0 ( appendix 22)
  • Polypropylene tube

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

  Materials
  • 1 × 109 cell/ml bacterial culture containing DNA of interest or 0.1 to 1 mg/ml DNA to be purified (5 to 10 µg DNA total)
  • 6 M sodium iodide (NaI) solution (filter through filter paper, store up to 3 months in the dark at 4°C)
  • recipeResuspension buffer (see recipe)
  • Lysis solution: 0.2 M NaOH/1.0% (w/v) SDS (store indefinitely at room temperature)
  • recipeNeutralization/binding solution (see recipe)
  • recipeWash buffer (see recipe)
  • TE buffer, pH 8.5 ( appendix 22) or nuclease‐free H 2O
  • Silica membrane spin columns (e.g., Qiagen, Promega, Invitrogen, Novagen)
  • 1.5‐ml microcentrifuge tubes

Alternate Protocol 2: DNA Purification Using Silica Membrane Spin Columns

  • 4 M ammonium acetate, pH 4.8
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

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.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library