Nucleic Acid Blotting: Southern and Northern

Laura L. Mays Hoopes1

1 Pomona College, Claremont, California
Publication Name:  Current Protocols Essential Laboratory Techniques
Unit Number:  Unit 8.2
DOI:  10.1002/9780470089941.et0802s6
Online Posting Date:  September, 2012
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Abstract

E.M. Southern invented blotting of DNA in 1975; the method was extended to RNA and named northern blotting in 1977. Southern blotting includes limited depurination, denaturation, and neutralization of the DNA in gels (where they have been separated in size by electrophoresis) and capillary transfer of the DNA onto nitrocellulose or nylon blotting membranes. For northern blotting, RNA is guarded from basic pH and RNase, denatured, separated by electrophoresis, and then blotted on to nylon blotting membranes. Either type of blot is then blocked to prevent nonspecific binding, hybridized with probe, and washed. Next, the sequences of interest are located by detecting labeled probes. One alternative method involves dot/slot blotting when the size of the nucleic acid being probed is not of interest. Also, electrophoretic transfer from polyacrylamide gels can be used when the nucleic acid fragments of interest are too small to be resolved on agarose gels. Artifacts in Southern blot can result from incomplete digestion, overloading the blotting membrane, incomplete blocking, damaged blot media, and air bubbles. In northern blotting, RNA quality must be monitored, and RNA that is degraded or contaminated with excess DNA should be avoided. Curr. Protoc. Essential Lab. Tech. 6:8.2.1‐8.2.26. © 2012 by John Wiley & Sons, Inc.

Keywords: RNA blotting; DNA blotting; electrophoresis; capillary transfer; hybridization; vacuum blotting; dot blotting

     
 
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Table of Contents

  • Overview and Principles
  • Strategic Questions
  • Strategic Planning
  • Safety Considerations
  • Protocols
  • Basic Protocol 1: Southern Blotting
  • Basic Protocol 2: Northern Blotting
  • Support Protocol 1: Assembling a Blotting Transfer Apparatus
  • Alternate Protocol 1: Acrylamide Gel Vacuum Blotting
  • Reagents and Solutions
  • Understanding Results
  • Troubleshooting
  • Variations
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Southern Blotting

  Materials
  • Agarose gel containing electrophoretically separated DNA fragments that have been stained, photographed, and destained (see unit 7.2; also see Voytas, )
  • Depurinator: 0.25 M HCl (store up to 6 months at room temperature in an air‐tight bottle)
  • Distilled water
  • Denaturant: 1.5 M NaCl/0.5 M NaOH (store up to 6 months at room temperature)
  • Neutralizer: 1.5 M NaCl/0.5 M Tris⋅Cl, pH 7.0 (store up to 6 months at room temperature) 2× and 20× SSC (see unit 3.3)
  • Prehybridization buffer with or without 50% formamide (see reciperecipes), 42°C or 68°C
  • Double‐ or single‐stranded probes (appropriate to the experiment; see unit 8.4)
  • Hybridization buffer with or without formamide (depending on hybridization temperature; see reciperecipes)
  • 2× SSC (see unit 3.3) containing 0.5% (w/v) and 0.1% (w/v) SDS
  • 0.1× SSC (see unit 3.3)/0.5% SDS
  • Glass dishes (e.g., Pyrex baking pans) for washing gels
  • Rocking or gyrating platform with low speed setting
  • Gloves
  • 10‐ or 25‐ml glass pipet
  • Plastic wrap
  • Whatman 3MM filter paper (Whatman, cat. no. 3030‐6189; 0.34 mm thickness)
  • Paper towels
  • 500‐ml flask
  • UV cross‐linker or UV transilluminator with 254‐nm wavelength light (for nylon membranes), or vacuum oven set to 80°C (for nitrocellulose membranes)
  • Reclosable plastic bags (larger than the membranes)
  • Hybridization bottles (or sealable plastic bag)
  • Hybridization oven (or water bath), set to 42°C or 68°C
  • 15‐ml disposable, sterile test tube
  • Long forceps
  • Additional reagents and equipment for assembling the blotting transfer apparatus ( protocol 3)
CAUTION: Wear gloves to protect hands from denaturant and to protect the membrane from contamination.

Basic Protocol 2: Northern Blotting

  Materials
  • DEPC‐treated water (see unit 3.3)
  • Agarose gel containing electrophoretically separated RNA fragments that have been stained, photographed, and destained (see unit 7.2; also see Voytas, )
  • 6× SSC treated with DEPC (see unit 3.3)
  • Prehybridization buffer (see recipe), 42°C
  • Double‐ or single‐stranded probes (appropriate to the experiment; see unit 8.4)
  • Hybridization buffer with or without formamide (depending on hybridization temperature; see reciperecipes)
  • 1× SSC/0.1% SDS treated with DEPC (see unit 3.3)
  • 0.2× SSC/0.1% SDS treated with DEPC (see unit 3.3), 68°C
  • Glass dishes (e.g., Pyrex baking pans), RNase free
  • Rocking or gyrating platform with low speed setting
  • Whatman 3MM filter paper (Whatman, cat. no. 3030‐6189; 0.34 mm thickness)
  • 10‐ to 25‐ml glass pipet, RNase free
  • Gloves
  • Plastic wrap
  • Paper towels
  • 500‐ml flask
  • UV cross‐linker or UV transilluminator with 254‐nm wavelength light (for nylon membranes), or vacuum oven set to 80°C (for nitrocellulose membranes)
  • Reclosable plastic bags (larger than the membranes)
  • 15‐ml sterile disposable plastic test tube
  • Hybridization bottles
  • Hybridization oven, set to 42°C or 68°C
  • Long forceps
  • Additional reagents and equipment for assembling the blotting transfer apparatus ( protocol 3)

Support Protocol 1: Assembling a Blotting Transfer Apparatus

  Materials
  • Absolute ethanol
  • Distilled water, sterile (treated with DEPC and then autoclaved for northern blots)
  • 20× (for nylon membranes) or 2× (for nitrocellulose membranes) SSC (see unit 3.3)
  • Scissors
  • Gloves
  • Whatman 3MM filter paper (Whatman #3030‐6189; 0.34 mm thickness)
  • Small glass or plastic dishes for hydrating membranes
  • Sponge, slightly larger than the gel being blotted
  • Uncharged nylon membrane (e.g., Hybond‐N, Amersham; (Duralon‐UV, Stratagene; or NEN GeneScreen, Dupont) or nitrocellulose membrane (for Southern blot only; e.g., BA 83 or BA 95, Schleicher & Schuell; Hybond‐C, Amersham; or Biodyne A, Pall), cut to fit gel (see unit 8.1 for overview of membrane types)
  • Large glass dish for transfer (to hold enough liquid to keep from going dry during an overnight capillary transfer period)

Alternate Protocol 1: Acrylamide Gel Vacuum Blotting

  Materials
  • Acrylamide gel containing electrophoretically separated nucleic acids (see Voytas, ) that have been stained, photographed, and destained, if desired
  • 0.5× TBE electrophoresis buffer: prepared in DEPC‐treated water and sterilized by passing through a 0.22‐µm filter (see unit 7.2), 4°C
  • Whatman 3MM filter paper (Whatman #3030‐6189; 0.34 mm thickness)
  • Uncharged nylon blotting membrane (e.g., Hybond‐N, Amersham; (Duralon‐UV, Stratagene; or NEN GeneScreen, Dupont)
  • Scotch‐Brite pads (supplied with Trans‐Blot)
  • 10‐ml pipet, sterile
  • Trans‐Blot electrophoresis cell (Bio‐Rad)
  • Blocks of ice frozen in plastic containers (containers supplied with Trans Blot)
  • Additional reagents and equipment for cross‐linking by UV irradiation **(see Basic Protocols protocol 11 and protocol 22)
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Figures

Videos

Literature Cited

   Alwine, J.C., Kemp, D.J., and Stark, G.R. 1977. Method for detection of specific RNAs in agarose gels by transfer to diazobenzylmethoxymethyl‐paper and hybridization with DNA probes. Proc. Natl. Acad. Sci. U.S.A. 74:5350‐5354.
   Boyle, A. and Perry‐O'Keefe, H. 1992. Labeling and colorimetric detection of nonisotopic probes. Curr. Protoc. Mol. Biol. 20:3.18.1‐3.18.9.
   Brown, T. 2004. Analysis of DNA sequences by blotting and hybridization. Curr. Protoc. Mol. Biol. 68:2.9.1‐2.9.20.
   Brown, T., Mackey, K., and Du, T. 2004. Analysis of RNA by northern and slot blot hybridization. Curr. Protoc. Mol. Biol. 67:4.9.1‐4.9.19.
   Ferriera, E.N., Galante, P.A., Carraro, D.N., and deSouza, S.J. 2007. Alternative splicing: A bioinformatic perspective. Mol. Biosyst. 3:473‐477.
   Perry‐O'Keefe, H. and Kissinger, C.M. 2000. Chemiluminescent detection of nonisotopic probes. Curr. Protoc. Mol. Biol. 26:3.19.1‐3.19.8.
   Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. Analysis of genomic DNA by Southern hybridization and northern hybridization In Molecular Cloning: A Laboratory Manual. 2nd ed. (J. Sambrook, E.F. Fritsch, and T. Maniatis, eds.) pp. E21‐E25. Cold Spring Harbor Press, Cold Spring Harbor, New York.
   Southern, E.M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503‐517.
   Sullivan, K.M. and Lilley, D.M.J. 1988. Helix stability and the mechanism of cruciform extrusion in supercoiled DNA. Nucleic Acids Res. 16:1079‐1093.
   Voytas, D. 2000. Agarose gel electrophoresis. Curr. Protoc. Mol. Biol. 51:2.5A.1‐2.5A.9.
Key References
   Alwine et al., 1977. See above.
  This paper is the classic description of northern blotting, and although it recommends a membrane that is no longer used, it clearly describes why this technique was needed and how to perform the transfer.
   Southern, 1975. See above.
  This paper contains the original description of Southern blotting and describes the reasons for its superiority to the methods previously available.
Internet Resources
   http://www.dnalc.org/ddnalc/resources/shockwave/southan.html
  Forensics Southern blot Internet animation requiring Shockwave software; leads user to identification of suspect.
   http://oba.od.nih.gov/rdna/rdna.html
  Provides access to government safety regulations for recombinant DNA experiments, including those using dangerous viruses.
   http://www.msdssearch.com
  A national repository of Materials Safety Data Sheets (MSDSs).
   http://hazard.com/msds
  Provide material safety data sheets for proper safe use and disposal of chemicals such as ethidium bromide, formaldehyde, and formamide that are used in these procedures.
   http://www.ambion.com/techlib/tb/tb_178.html
  Ambion Web site; RNA reagents and consumables.
   http://www.accessexcellence.org/RC/VL/GG/ecb/southern_blotting.html
  Southern blotting explained on Access Excellence, the National Health Museum Resource Center.
   http://www.bio.davidson.edu/COURSES/GENOMICS/method/Southernblot.html
  An explanation of Southern blotting by A. Malcolm Campbell (related to his genomics class) on the Davidson College Biology Department's home page.
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