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Nucleic Acid Blotting: Southern and Northern

Laura L. Mays Hoopes¹

¹Pomona College, Claremont, California

Publication Name:

Current Protocols Essential Laboratory Techniques

Unit Number:

Unit 8.2

DOI:

10.1002/9780470089941.et0802s00

Online Posting Date:

October, 2008

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Abstract

E.M. Southern invented blotting of DNA in 1975; the method was extended to RNA in 1977. Standard 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 base and RNase, denatured, separated by electrophoresis, and then blotted to nylon blotting membranes. Both types of blots are then blocked to prevent nonspecific binding, hybridized with probe, and washed. Next the sequences of interest are located by detecting labeled probes. Alternative methods involve dot/slot blotting when the size of the nucleic acid being probed is not of interest. Electrophoretic transfer from polyacrylamide gels can be used when the nucleic acid fragments of interest are too small to be effectively 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.

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

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Overview and Principles
Strategic Planning
Safety Considerations
Protocols
Basic Protocol 1: Southern Blotting
Basic Protocol 2: Northern Blotting
Support Protocol: Assembling a Blotting Transfer Apparatus
Alternate Protocol: 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)
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 recipes), 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 recipes)
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
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 (Support Protocol)

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)
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 recipes)
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
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 (Support Protocol)

Support Protocol: 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 sheets
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 and 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: Acrylamide Gel Vacuum Blotting

Materials

Acrylamide gel containing electrophoretically separated nucleic acids (see CP Molecular Biology Unit 2.5A; Voytas, 2000) 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
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 Protocol 1 or 2)

Looking for materials? Suppliers Guide

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Figures

Figure 8.2.1

Apparatus for Southern blotting. Two alternative transfer setups for upward capillary transfer are shown: (A) sponge method and (B) Whatman 3MM filter paper wick method. Originally published in CP Molecular Biology Unit 2.9 (Brown, 2004).

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Figure 8.2.2

Problems in Southern blotting. (A) A good Southern blot, showing low background and sharp bands. (B) Excessive DNA (right lanes) and DNA incompletely digested by restriction enzymes (left lanes) can obscure Southern blotting results. Compare the excessive DNA at the top of the right lanes to the two bands of DNA in the left lanes, produced by completely digested DNA. (C) Effects of inadequate blocking on blots; note the edges of the blot have nonspecific label stuck on them. (D) Effects of damage to the blot medium on images. Note the cracks with label stuck in them, the results of damage to the blot membrane when inserting it into a hybridization tube. (E) Effect of a bubble between the gel and the blot medium causes a “pock” where no transfer occurs, making a white circular artifact on the blot.

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Figure 8.2.3

Saccharomyces cerevisiae RNA quality control gel with problems identified. Lane 1: Standards of double stranded DNA. Lane 2: RNA sample with mostly small RNA, possibly transfer RNA plus degraded fragments. Lane 3: RNA sample with some DNA (compare with lane 4), very light bands for the two large rRNAs (two bands running just ahead of the DNA). Lane 4: RNA sample that is almost all genomic DNA. Lane 5: Degraded RNA that runs as a smear and lacks distinct bands for the larger rRNAs. Lane 6: Good sample of RNA that has strong rRNA bands, not too much DNA or tRNA. Lane 7: Sample of RNA with excessive DNA so it may hybridize badly with probes. Lane 8: The rRNA bands are strong but the small RNA is excessive, suggesting degradation.

View Image

Literature Cited

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. 68:2.9.1-2.8.20.
	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. pp. E21-E25. Cold Spring Harbor Press, Cold Spring Harbor, N.Y.
	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. Nucl. 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, 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://www4.od.nih.gov/oba/rac/aboutrdagt.htm
Provides access to government safety regulations for recombinant DNA experiments, including those using dangerous viruses.
	http://www.msdssearch.com
	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
Explains the use and misuse of DEPC in treating solutions to remove RNase activity.
	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.