Southern Blotting

Terry Brown1

1 University of Manchester Institute of Science and Technology, Manchester, United Kingdom
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 2.9A
DOI:  10.1002/0471142727.mb0209as21
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 (the properties and advantages of the different types of membrane, transfer buffer, and transfer method are discussed in detail), 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 presents several protocols for DNA transfer to nylon and nitrocellulose membranes, and for calibrating a UV transilluminator to cross‐link the transferred DNA to the membrane.

     
 
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Table of Contents

  • Analysis of DNA Sequences By Blotting and Hybridization
  • 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
  • Alternate Protocol 3: Electroblotting from a Polyacrylamide Gel to a Nylon Membrane
  • Commentary
  • 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)
  • 20× and 2× SSC ( appendix 22)
  • Oblong sponge slightly larger than the gel being blotted
  • Whatman 3MM filter paper sheets
  • Nylon or nitrocellulose membrane (see Table 2.9.1 for suppliers)
    Table 2.9.1   Materials   Properties of Materials used for Immobilization of Nucleic Acids a   Properties of Materials used for Immobilization of Nucleic Acids

    Nitrocellulose Supported nitrocellulose Uncharged nylon Positively charged nylon Activated papers
    Application ssDNA, RNA, protein ssDNA, RNA, protein ssDNA, dsDNA, DNA, protein ssDNA, dsDNA, RNA, protein ssDNA, RNA
    Binding capacity (µg nucleic acid/cm2) 80‐100 80‐100 400‐600 400‐600 2‐40
    Tensile strength Poor Good Good Good Good
    Mode of nucleic acid attachment b Noncovalent Noncovalent Covalent Covalent Covalent
    Lower size limit for efficient nucleic acid retention 500 nt 500 nt 50 nt or bp 50 nt or bp 5 nt
    Suitability for reprobing Poor (fragile) Poor (loss of signal) Good Good Good
    Commercial examples Schleicher & Schuell BA83, BA85; Amersham Hybond‐C; PALL Biodyne A Schleicher & Schuell BA‐S; Amersham Hybond‐C extra Amersham Hybond‐N; Stratagene Duralon‐UV; Du Pont NEN GeneScreen Schleicher & Schuell Nytran; Amersham Hybond‐N+; Bio‐Rad ZetaProbe; PALL Biodyne B; Du Pont NEN GeneScreen Plus Schleicher & Schuell APT papers

     aThis table is based on Brown ( ), with permission from BIOS Scientific Publishers Ltd.
     bAfter suitable immobilization procedure (see text).
  • UV‐transparent plastic wrap (e.g., Saran Wrap) for nylon membranes
  • UV transilluminator (unit 2.5) or UV light box (e.g., Stratagene Stratalinker) for nylon membranes
  • Additional reagents and equipment for restriction endonuclease digestion (unit 3.1) and agarose gel electrophoresis (unit 2.5)
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
  • Additional reagents and equipment for DNA dot blotting (unit 2.9) and hybridization analysis (unit 2.10)
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
  • 0.4 M (for charged membrane) or 0.25 M (for uncharged membrane) NaOH
  • 0.25 M NaOH/ 1.5 M NaCl for uncharged membrane
  • Positively charged or uncharged nylon membrane (see Table 2.9.1 for suppliers)
CAUTION: Wear gloves to protect your hands from the acid and alkali solutions and to protect the membrane from contamination.

Alternate Protocol 2: Southern Blotting by Downward Capillary Transfer

  Additional Materials
  • 0.5× TBE electrophoresis buffer ( appendix 22)
  • Scotch‐Brite pads (supplied with Trans‐Blot apparatus)
  • Trans‐Blot electroblotting cell (Bio‐Rad) with cooling coil, or other electroblotting apparatus (unit 10.8)
  • Additional reagents and equipment for nondenaturing (unit 2.7) or denaturing (unit 2.12) polyacrylamide gel electrophoresis and for electroblotting (unit 10.8)
CAUTION: Wear gloves to protect the membrane from contamination.
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Figures

  •   FigureFigure 2.9.1 Two alternative transfer pyramid setups for Southern blotting via upward capillary transfer. (A) Sponge method. (B) Whatman 3MM filter paper wick method.
  •   FigureFigure 2.9.2 Transfer pyramid setup for Southern blotting via downward capillary transfer. Adapted with permission from Academic Press.

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.
  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.
   Doel, M.T., Houghton, M., Cook, E.A., and Carey, N.H. 1977. The presence of ovalbumin mRNA coding sequences in multiple restriction fragments of chicken DNA. Nucl. Acids Res. 4:3701‐3713.
   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.
   Hall, B.D. and Spiegelman, S. 1961. Sequence complementarity of T2‐DNA and T2‐specific RNA. Proc. Natl. Acad. Sci. U.S.A. 47:137‐146.
   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.
   Lichtenstein, A.V., Moiseev, V., and Zaboikin, M.M. 1990. A procedure for DNA and RNA transfer to membrane filters avoiding weight‐induced gel flattening. Anal. Biochem. 191:187‐191.
   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.
   Noyes, B.E. and Stark, G.R. 1975. Nucleic acid hybridization using DNA covalently coupled to cellulose. Cell 5:301‐310.
   Nygaard, A.P. and Hall, B.D. 1963. A method for the detection of RNA‐DNA complexes. Biochem. Biophys. Res. Commun. 12:98‐104.
   Peferoen, M., Huybrechts, R., and De Loof, A. 1982. Vacuum‐blotting: A new simple and efficient transfer of proteins from sodium dodecyl sulfate–polyacrylamide gels to nitrocellulose. FEBS Letts. 145:369‐372.
   Reed, K.C. and Mann, D.A. 1985. Rapid transfer of DNA from agarose gels to nylon membranes. Nucl. Acids Res. 13:7207‐7221.
   Seed, B. 1982. Diazotizable arylamine cellulose papers for the coupling and hybridization of nucleic acids. Nucl. Acids Res. 10:1799‐1810.
   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.
Key Reference
   Southern, 1975. See above.
   First description of capillary transfer from gel to membrane.
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