Preparation, Restriction, and Hybridization Analysis of Mammalian Genomic DNA for Pulsed-Field Gel Electrophoresis

The aim of this chapter is to provide a very practical set of instructions that will enable workers with some experience in standard DNA preparation and electrophoresis techniques to prepare and analyze mammalian DNA by pulsed-field gel electrophoresis (PFGE; see also refs. 1 and 2 for additional PFGE protocols). Detailed methods are provided that describe: sources of mammalian DNA, preparation of DNA, restriction enzyme digests, standard PFGE running conditions, preparation of size markers, and preparation, hybridization, and analysis of blots. Notes about potential pitfalls accompany each section together with a trouble-shooting guide in Section 5 .