Southern Blotting

Terry Brown1

1 University of Manchester Institute of Science and Technology, Manchester, United Kingdom
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 10.6A
DOI:  10.1002/0471142735.im1006as06
Online Posting Date:  May, 2001
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Abstract

Southern blotting is the transfer of DNA fragments from an electrophoresis gel to a membrane support, resulting in immobilization of the DNA fragments, so the membrane carries a semipermanent reproduction of the banding pattern of the gel. After immobilization, the DNA can be subjected to hybridization analysis, enabling bands with sequence similarity to a labeled probe to be identified. This unit describes Southern blotting via upward capillary transfer of DNA from an agarose gel onto a nylon or nitrocellulose membrane, and subsequent immobilization by UV irradiation (for nylon) or baking (for nitrocellulose). A describes how to calibrate a UV transilluminator for optimal UV irradiation of a nylon membrane. An alternate protocol details transfer using nylon membranes and an alkaline buffer, and is primarily used with positively charged nylon membranes. A second alternate protocol describes a transfer method based on a different transfer‐stack setup. The traditional method of upward capillary transfer of DNA from gel to membrane has certain disadvantages, notably the fact that the gel can become crushed by the weighted filter papers and paper towels that are laid on top of it. This slows down the blotting process and may reduce the amount of DNA that can be transferred. The downward capillary method described in the second alternate protocol is therefore more rapid and can result in more complete transfer.

     
 
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Table of Contents

  • Analysis of DNA Sequences
  • Basic Protocol 1: Southern Blotting onto a Nylon or Nitrocellulose Membrane with High‐Salt Buffer
  • Support Protocol 1: Calibration of a UV Transilluminator
  • Alternate Protocol 1: Southern Blotting onto a Nylon Membrane with an Alkaline Buffer
  • Alternate Protocol 2: Southern Blotting by Downward Capillary Transfer
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Southern Blotting onto a Nylon or Nitrocellulose Membrane with High‐Salt Buffer

  Materials
  • DNA samples to be analyzed
  • 0.25 M HCl
  • Denaturation solution: 1.5 M NaCl/ 0.5 M NaOH (store at room temperature)
  • Neutralization solution: 1.5 M NaCl/ 0.5 M Tris⋅Cl, pH 7.0 (store at room temperature)
  • recipe20× and 2× SSC
  • Oblong sponge slightly larger than the gel being blotted
  • Whatman 3MM filter paper sheets
  • Nylon or nitrocellulose membrane (see Table 10.6.1 for suppliers)
  • UV‐transparent plastic wrap (e.g., Saran Wrap) for nylon membranes
  • UV transilluminator (unit 10.6) or UV light box (e.g., Stratagene Stratalinker) for nylon membranes
  • Additional reagents and equipment for restriction endonuclease digestion (unit 10.8) and agarose gel electrophoresis (unit 10.4)
CAUTION: Wear gloves from step of the protocol onward to protect your hands from the acid and alkali solutions and to protect the membrane from contamination.

Support Protocol 1: Calibration of a UV Transilluminator

  Additional Materials
  • DNA probe labeled to a specific activity of 108 dpm/µg (unit 10.9)
  • 6× SSC
  • Additional reagents and equipment for hybridization analysis (unit 10.6)
CAUTION: Exposure to UV irradiation is harmful to the eyes and skin. Wear suitable eye protection and avoid exposure of bare skin.

Alternate Protocol 1: Southern Blotting onto a Nylon Membrane with an Alkaline Buffer

  Additional Materials
  • NaOH: 0.4 M (for charged membrane) or 0.25 M (for uncharged membrane)
  • 0.25 M NaOH/ 1.5 M NaCl for uncharged membrane
  • Positively charged or uncharged nylon membrane (see Table 10.6.1 for suppliers)
CAUTION: Wear gloves to protect your hands from the acid and alkali solutions and to protect the membrane from contamination.
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Figures

Videos

Literature Cited

Literature Cited
   Bittner, M., Kupferer, P., and Morris, C.F. 1980. Electrophoretic transfer of proteins and nucleic acids from slab gels to diazobenzyloxymethyl cellulose or nitrocellulose sheets. Anal. Biochem. 102:459‐471.
   Boyle, A. and Perry‐O'Keefe, H. 1992. Labeling and colorimetric detection of nonisotopic probes. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston D.D. Moore, J.G. Seidman J.A. Smith, and K. Struhl (eds.) pp. 3.18.1‐3.18.9. Greene Publishing and Wiley‐Interscience. New York.
  Brown, T.A. (ed.) 1991. Molecular Biology Labfax. BIOS Scientific Publishers, Oxford.
   Chomczynski, P. 1992. One‐hour downward alkaline capillary transfer for blotting of DNA and RNA. Anal. Biochem. 201:134‐139.
  Davies, K.E. (ed.) 1986. Human Genetic Diseases: A Practical Approach. IRL Press at Oxford University Press, Oxford.
   Dyson, N.J. 1991. immobilization of nucleic acids and hybridization analysis. In Essential Molecular Biology: A Practical Approach, Vol. 2 (T.A. Brown, ed.) pp. 111‐156. IRL Press at Oxford University Press, Oxford.
   Haas, M., Vogt, M., and Dulbecco, R. 1972. Loss of simian virus 40 DNA‐RNA hybrids from nitrocellulose membrane: Implications for the study of virus‐host interactions. Proc. Natl. Acad. Sci. U.S.A. 69:2160‐2164.
   Jeffreys, A.J., Wilson, V., and Thein, S.L. 1985. Hypervariable ‘minisatellite’ regions in human DNA. Nature 314:67‐73.
   Khandjian, E.W. 1987. Optimized hybridization of DNA blotted and fixed to nitrocellulose and nylon membranes. Bio/Technology 5:165‐167.
   Li, J.K., Parker, B., and Kowalic, T. 1987. Rapid alkaline blot‐transfer of viral dsRNAs. Anal. Biochem. 163:210‐218.
   Nagamine, Y., Sentenac, A., and Fromageot, P. 1980. Selective blotting of restriction DNA fragments on nitrocellulose membranes at low salt concentrations. Nucl. Acids Res. 8:2453‐2460.
   Perry‐O'Keefe, H. and Kissinger, C.M. 1992. Chemiluminescent detection of nonisotopic probes. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 3.19.1‐3.19.8. Greene Publishing and Wiley‐Interscience, New York.
   Reed, K.C. and Mann, D.A. 1985. Rapid transfer of DNA from agarose gels to nylon membranes. Nucl. Acids Res. 13:7207‐7221.
   Smith, G.E. and Summers, M.D. 1980. The bidirectional transfer of DNA and RNA to nitrocellulose or diazobenzyloxymethyl paper. Anal. Biochem. 109:123‐129.
   Smith, M.R., Devine, C.S., Cohn, S.M., and Lieberman, M.W. 1984. Quantitative electrophoretic transfer of DNA from polyacrylamide or agarose gels to nitrocellulose. Anal. Biochem. 137:120‐124.
   Southern, E.M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503‐517.
   Stellwag, E.J. and Dahlberg, A.E. 1980. Electrophoretic transfer of DNA, RNA and protein onto diazobenzyloxymethyl (DBM) paper. Nucl. Acids Res. 8:299‐317.
   Towbin, H., Staehelin, T., and Gordon, J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applicatons. Proc. Natl. Acad. Sci. U.S.A. 76:4350‐4354.
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