Southern Blotting

Terry Brown1

1 University of Manchester Institute of Science and Technology, Manchester, null
Publication Name:  Current Protocols in Protein Science
Unit Number:  Appendix 4G
DOI:  10.1002/0471140864.psa04gs13
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 appendix describes Southern blotting via upward capillary transfer of DNA from an agarose gel onto a nylon or nitrocellulose membrane, using a high‐salt transfer buffer to promote binding of DNA to the membrane. With the high‐salt buffer, the DNA becomes bound to the membrane during transfer but not permanently immobilized. Immobilization is achieved 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. The advantage of this combination is that no post‐transfer immobilization step is required, as the positively charged membrane binds DNA irreversibly under alkaline transfer conditions. The method can also be used with neutral nylon membranes but less DNA will be retained. 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 described in the first basic and alternate protocols 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 than the basic protocol and can result in more complete transfer. Although the ease and reliability of capillary transfer methods makes this far and away the most popular system for Southern blotting with agarose gels, it unfortunately does not work with polyacrylamide gels, whose smaller pore size impedes the transverse movement of the DNA molecules. The third alternate protocol describes an electroblotting procedure that is currently the most reliable method for transfer of DNA from a polyacrylamide gel. Dot and slot blotting are also described.

     
 
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Table of Contents

  • 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
  • Basic Protocol 2: Dot and Slot Blotting of DNA onto Uncharged Nylon and Nitrocellulose Membranes Using a Manifold
  • Alternate Protocol 4: Dot and Slot Blotting of DNA onto a Positively Charged Nylon Membrane Using a Manifold
  • Alternate Protocol 5: Manual Preparation of a DNA Dot Blot
  • 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
  • 0.25 M HCl ( appendix 2E)
  • 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 2E)
  • Nylon or nitrocellulose membrane (see Table 4.0.1 or suppliers)
  • UV transilluminator or UV light box (e.g., Stratagene Stratalinker) for nylon membranes
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. Handle the membrane with blunt‐end forceps.
Table 0.g.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 RNA, 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 (1991), with permission from BIOS Scientific Publishers Ltd.
 bAfter suitable immobilization procedure (see text)

Support Protocol 1: Calibration of a UV Transilluminator

  • 0.4 M NaOH (for charged membrane) or 0.25 M NaOH (for uncharged membrane)
  • 0.25 M NaOH/1.5 M NaCl for uncharged membrane
  • Positively charged or uncharged nylon membrane (see Table 4.0.1 for suppliers)

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

  • 0.5× TBE electrophoresis buffer ( appendix 2E)
  • Scotch‐Brite pads (supplied with Trans‐Blot apparatus)
  • Trans‐Blot electroblotting cell (Bio‐Rad) with cooling coil, or other electroblotting apparatus (unit 10.7)

Alternate Protocol 2: Southern Blotting by Downward Capillary Transfer

  Materials
  • 6× and 20× SSC ( appendix 2E)
  • Denaturation solution: 1.5 M NaCl/0.5 M NaOH (store at room temperature)
  • Neutralization solution: 1 M NaCl/ 0.5 M Tris⋅Cl, pH 7.0 (store at room temperature)
  • Uncharged nylon or nitrocellulose membrane (see Table 4.0.1)
  • Dot/slot blotting manifold (e.g., Bio‐Rad Bio‐Dot SF or Schleicher and Schuell Minifold II)
  • UV transilluminator for nylon membranes

Alternate Protocol 3: Electroblotting from a Polyacrylamide Gel to a Nylon Membrane

  • Positively charged nylon membrane (see Table 4.0.1)
  • 0.4 M and 1 M NaOH ( appendix 2E)
  • 200 mM EDTA, pH 8.2 ( appendix 2E)
  • 2× SSC ( appendix 2E)
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Figures

  •   FigureFigure a0.4G.1 Transfer stacks for upward capillary transfer. (A) Sponge method. (B) Filter paper wick method.
  •   FigureFigure a0.4G.2 Transfer pyramid for downward capillary transfer. Adapted with permission from Academic Press.
  •   FigureFigure a0.4G.3 Dot (left) and slot (right) blot manifold architectures.

Videos

Literature Cited

Literature Cited
   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 Oxford
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