Purification of DNA by Anion‐Exchange Chromatography

Kim Budelier1, Joachim Schorr2

1 QIAGEN, Inc., Valencia, California, 2 QIAGEN GmbH, Hilden, Germany
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 2.1B
DOI:  10.1002/0471142727.mb0201bs42
Online Posting Date:  May, 2001
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Abstract

Column chromatography has evolved to provide a rapid and effective alternative to more laborious methods for preparing high‐quality DNA, such as CsCl‐gradient centrifugation. This unit describes the use of a column made of a unique anion‐exchange resin that selectively binds nucleic acids, allowing rapid separation of DNA from contaminating RNA, proteins, carbohydrates, and metabolites. The procedure employs columns supplied by QIAGEN; other preparation methods are available from other suppliers. A crude nucleic acid sample (usually a cleared cell lysate) is applied to the QIAGEN tip under conditions that favor binding. Contaminants in the sample are washed from the column with a moderate‐salt buffer, and DNA is eluted using a high‐salt buffer.

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1:

  Materials
  • Plasmid‐ or phage‐bearing bacterial culture or mammalian, plant, or bacterial cell culture
  • recipeBuffer QBT (equilibration buffer; see recipe)
  • QIAGEN‐tip of appropriate size (Table 2.1.1)
    Table 2.1.1   MaterialsRecommended Loading Volumes, DNA Capacities, and Culture Volumes for QIAGEN‐tips

    QIAGEN‐tip 100   QIAGEN‐tip 500   QIAGEN‐tip 2500   QIAGEN‐tip 10000 
    Column DNA capacity
    Plasmid or ds phage 100 µg 500 µg 2500 µg 10000 µg
    Cosmid or λ phage 60 µg 300 µg 1500 µg 6000 µg
    Mammalian or B. subtilis genomic 80 µg 400 µg 2000 µg 8000 µg
    Culture volume for plasmid DNA
    High‐copy‐number plasmid 25 ml 100 ml 500 ml 2.5 liters
    Low‐copy‐number plasmid 100 ml 500 ml 2.5 liters 5 liters
    Culture volume for genomic DNA
    B. subtilis 8 ml 40 ml 200 ml 800 ml
    Mammalian (no. cells) 2 × 107 1 × 108 5 × 108 2 × 109

  • recipeBuffer QC (washing buffer; see recipe)
  • recipeBuffer QF (eluting buffer; see recipe)
  • TE buffer, pH 8.0 ( appendix 22)
  • Isopropanol, room temperature
  • 70% (v/v) ethanol, ice cold
  • Beckman JS‐13, Sorvall HB‐4 or HB‐6, or equivalent rotor
  • Additional reagents and equipment for preparation of mammalian, plant, or bacterial genomic DNA (cell lysate; units 2.2 2.4), alkaline lysis preparation of plasmid DNA (unit 1.7), or preparation of phage lysates (unit 1.13)
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Figures

Videos

Literature Cited

Literature Cited
   Chen, W., Andres, D., Goldstein, J., Russell, D., and Brown, M. 1991. cDNA cloning and expression of the peptide‐binding beta subunit of Rat p21ras farnesyltransferase, the counterpart of yeast DPR1/RAM1. Cell 66:327‐334.
   Ehlert, F., Bierbaum, P., and Schorr, J. 1993. Importance of DNA quality for transfection efficiency. BioTechniques 14:546.
   Hall, B., Betts, P., and Wootton, J. 1989. DNA sequence analysis of artificially evolved ebg enzyme and ebg repressor gene. Genetics 123:635‐648.
   Jones, D. and Winistorfer, S. 1992. Recombinant circle PCR and recombinant PCR for site‐specific mutagenesis without PCR production purification. BioTechniques 12:528‐535.
   Jung, M., Dritschilo, A., and Kasid, U. 1992. Reliable and efficient direct sequencing of PCR double‐stranded genomic DNA template. PCR Methods Appl. 1:171‐174.
   Kayne, P., Kim, U., Han, M., Mullen, J., Yoshizaki, F., and Grunstein, M. 1988. Extremely conserved histone H4 N terminus is dispensable for growth but essential for repressing the silent mating loci in yeast. Cell 55:27‐39.
   Lutze, L. and Winegar, R.A. 1990. A quick and efficient method for the recovery of plasmid or viral DNA from mammalian cells. Nucl. Acids Res. 18:6150.
   Luytjes, W., Krystal, M., Enami, M., Parvin, J., and Palese, P. 1989. Amplification, expression, and packaging of a foreign gene by influenza virus. Cell 59:1108‐1113.
  QIAGEN. 1997. QIAGEN Plasmid Purification Handbook. QIAGEN, Valencia, Calif.
   Schleef, M. and Heimann, P. 1993. Cesium chloride or column preparation? An electron microscopical view of plasmid preparations. BioTechniques 14:544.
   Tartoff, K.D. and Hobbs, C.A. 1987. Improved media for growing plasmid and cosmid clones. Focus (BRL) 9:12.
   Voss, H., Zimmerman, J., Schwager, C., Stegemann, J., Erfle, H., Stuckey, K., and Ansorge, W. 1990. Automated fluorescent sequencing of cosmic DNA. Nucl. Acids Res. 18:1066.
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