2. Cutting the DNA into several pieces of different sizes. This is done using one or more restriction enzymes.
3. Sorting the DNA pieces by size. The process by which the size separation, "size fractionation," is done is called gel electrophoresis. The DNA is poured into a gel, such as agarose, and an electrical charge is applied to the gel, with the positive charge at the bottom and the negative charge at the top. Because DNA has a slightly negative charge, the pieces of DNA will be attracted towards the bottom of the gel; the smaller pieces, however, will be able to move more quickly and thus further towards the bottom than the larger pieces. The different-sized pieces of DNA will therefore be separated by size, with the smaller pieces towards the bottom and the larger pieces towards the top.
4. Denaturing the DNA, so that all of the DNA is rendered single-stranded. This can be done either by heating or chemically treating the DNA in the gel.
5. Blotting the DNA. The gel with the size-fractionated DNA is applied to a sheet of nitrocellulose paper, and then baked to permanently attach the DNA to the sheet. The Southern Blot is now ready to be analyzed.
In order to analyze a Southern Blot, a radioactive genetic probe is used in a hybridization reaction with the DNA in question (see next topics for more information). If an X-ray is taken of the Southern Blot after a radioactive probe has been allowed to bond with the denatured DNA on the paper, only the areas where the radioactive probe binds [red] will show up on the film. This allows researchers to identify, in a particular person's DNA, the occurrence and frequency of the particular genetic pattern contained in the probe.