Transferring Electrophoretically Separated DNA from Agarose Gels to a Membrane by Southern Blotting
To detect or probe for DNA containing complementary sequences to other DNA or RNA sequences, the DNA separated on the gel must first be transferred and immobilized on a solid support such as nitrocellulose or nylon membranes. The Southern blotting procedure, originally demonstrated by Southern in 1975, accomplishes this. In this procedure, the double-stranded DNA duplexes are depurinated with acid, then denatured by treatment with an alkali solution while still within the gel. Alkali treatment produces single-stranded DNA, which binds to the membrane while double-stranded DNA does not. The gel is neutralized and placed on top of a layer of filter paper (wick) soaked in a high-salt buffer. A membrane is then placed on the gel followed by a thin layer of filter paper on the membrane. This is followed by a stack of paper towels on top of which a weight is placed. This arrangement creates a moisture gradient and draws the high-salt buffer solution upwards by capillary action, through the filter paper, gel, and paper towels. This flow of buffer by capillary action transfers the DNA to the membrane, but large fragments and supercoiled plasmid DNA do not transfer efficiently. However, efficient transfer is achieved after the DNA is depurinated with acid and the depurinated sites cleaved by alkali treatment. On completing the transfer, the membrane is baked to bind (immobilize) the DNA, almost permanently, on the membrane. In this form, the immobilized DNA can be probed for specific DNA sequences of interest. In this experiment, supercoiled plasmid DNA or restriction endonuclease digested genomic DNA fragments separated by agarose electrophoresis are transferred onto nitrocellulose or nylon membranes by Southern blotting.
KeywordsFilter Paper Nylon Membrane Paper Towel Alkali Treatment Capillary Action
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- Maniatis, T., E.F. Fritsch, and J. Sambrook. 1982. Molecular cloning, pp. 382–389. In A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar