Separation of Small DNA Fragments by Conventional Gel Electrophoresis

Large amounts of small (<1000-bp) DNA fragments can be separated by conventional electrophoretic means. The purified fragments can then be used for cloning, sequencing, and labeling. In this unit, the techniques of DNA separation via both nondenaturing polyacrylamide and sieving agarose electrophoresis are discussed. Methods are detailed for the pouring and electrophoresis of nondenaturing polyacrylamide gels, followed by elution of the labeled or unlabeled separated DNA fragments from the gels by either passive diffusion or electroelution. A method is also provided for the use of sieving agarose, a specially treated type of agarose designed to be used at high concentrations. Poured and run like conventional agarose gels, this matrix can resolve small DNA fragments much like a nondenaturing polyacrylamide gel.