Isolation of Genomic DNA from Mammalian Cells

John R. Gilbert1, Jeffrey M. Vance1

1 Duke University Medical Center, Durham, North Carolina
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Appendix 3B
DOI:  10.1002/0471142905.hga03bs19
Online Posting Date:  May, 2001
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Abstract

This unit describes simple, cost‐effective preparation of DNA from whole blood or cultured cells that yields high‐molecular‐weight DNA suitable for both Southern blotting and the polymerase chain reaction. Preparation time may be shortened by substituting a high‐salt precipitation procedure for the dialysis step; however, this results in a smaller average fragment size. The isolation of DNA from buccal swabs, collected from the inside of the cheek, is also described. The DNA is suitable for PCR analysis. Preparation of buffered phenol for DNA extraction is described in a support protocol.This unit describes simple, cost‐effective preparation of DNA from whole blood or cultured cells that yields high‐molecular‐we

     
 
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Table of Contents

  • Basic Protocol 1: DNA Isolation from Whole Blood
  • Alternate Protocol 1: DNA Isolation from Cell Pellets
  • Alternate Protocol 2: Recovery of Genomic DNA by High‐Salt Precipitation
  • Basic Protocol 2: Isolation of DNA from Buccal Swabs
  • Support Protocol 1: Preparation of Buffered Phenol
  • Reagents and Solutions
  • Literature Cited
     
 
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Materials

Basic Protocol 1: DNA Isolation from Whole Blood

  Materials
  • 10 ml fresh or thawed frozen whole blood
  • recipeNKM buffer (see recipe), 4°C
  • recipeResuspension buffer (see recipe)
  • recipe10× TEN solution (see recipe)
  • recipe2 mg/ml proteinase K (see recipe)
  • 10% (w/v) SDS
  • Buffered phenol (see protocol 5)
  • 25:24:1 (v/v) phenol/chloroform/isoamyl alcohol ( appendix 3C)
  • 24:1 (v/v) chloroform/isoamyl alcohol
  • TE buffer, pH 8.0 ( appendix 2D)
  • recipeDialysis buffer (see recipe)
  • 65°C water bath
  • Dialysis tubing (MWCO 5000)
  • Additional reagents and equipment for quantitation of DNA by absorption spectroscopy ( appendix 3D)

Alternate Protocol 1: DNA Isolation from Cell Pellets

  • Cultured cells ( appendix 3G & )
  • Phosphate‐buffered saline (PBS; appendix 2D), sterile
  • recipeSucrose lysis solution (see recipe)
  • 50‐ml centrifuge tubes

Alternate Protocol 2: Recovery of Genomic DNA by High‐Salt Precipitation

  • 5 M NaCl
  • 100% and 70% ethanol

Basic Protocol 2: Isolation of DNA from Buccal Swabs

  Materials
  • 50 mM NaOH
  • 1 M Tris⋅Cl, pH 6.5 ( appendix 2D)
  • 70% ethanol
  • CYTO‐PAK with Cyto‐soft brush (CP‐5B; Medical Packaging) or Caliber Cotton‐Tipped Applicator swabs (size 6 in. L; Allegiance Healthcare)
  • 95°C heating block

Support Protocol 1: Preparation of Buffered Phenol

  Materials
  • Distilled phenol (store frozen at −20°C)
  • 1 M Tris⋅Cl, pH 7.5 ( appendix 2D)
  • 65°C water bath
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Figures

Videos

Literature Cited

Literature Cited
   Richards, B., Skoletsky, J., Shuber, A., Balfour, R., Stern, R., Dorkin, H., Parad, R., Witt, D., and Klinger, K. 1994. Multiplex PCR amplification from the CFTR gene using DNA prepared from buccal brushes/swabs. Hum. Mol. Genet. 2:159‐163.
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