Southern Blotting

Abstract

In Southern blotting, DNA is digested with one or more restriction enzymes, and the resulting fragments are separated according to size by electrophoresis through a standard agarose gel. The DNA is then denatured in situ and transferred from the gel to a solid support (usually a nylon or nitrocellulose membrane). The relative positions of the DNA fragments are preserved during their transfer to the membrane. The DNA is then fixed to the membrane and prepared for hybridization. Alternatively, DNA can be simultaneously transferred from the top and bottom surfaces of a single agarose gel to two membranes. This procedure is useful when the need arises to analyze the same set of restriction fragments with two different probes. Transfer of DNA fragments is rapid, but the efficiency is low because the agarose gel quickly becomes dehydrated as fluid is withdrawn from both sides. The method therefore works best when the target sequences are present in high concentration (e.g., when analyzing cloned DNAs [plasmids, bacteriophages, cosmids, PACs, or BACs] or less complex genomes [those of Saccharomyces cerevisiae or Drosophila]). Too little mammalian genomic DNA is transferred to allow signals from single-copy sequences to be detected in a reproducible or timely fashion.