Southern blotting is the transfer of DNA fragments from an electrophoresis gel to a membrane support. The transfer or a subsequent treatment results 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 hybridiza tion analysis, enabling bands with sequence similarity to a labeled probe to be identified.

When setting up a Southern transfer, choices must be made between different types of membrane, transfer buffer, and transfer method. The most popular membranes are made of nitrocellulose, uncharged nylon, or positively charged nylon. Although these materials have different properties, the three types of membrane are virtually interchangeable for many applications. The main advantage of nylon membranes is that they are relatively robust and so can be reprobed ten or more times before falling apart. Nitrocellulose membranes are fragile and can rarely be reprobed more than three times; however, these are still extensively used, as they give lower backgrounds with some types of hybridization probe. The properties and advantages of the different membranes are discussed more fully in the commentary.

The basic protocol 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 support protocol describes how to calibrate a UV transilluminator for optimal UV irradiation of a nylon membrane.

The first 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. The 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 polyacryl amide gel.