Indexed on: 01 Jan '92Published on: 01 Jan '92Published in: Methods in molecular biology (Clifton, N.J.)
The preparation and manipulation of very large DNA molecules for pulsed-field gel electrophoresis (PFGE) requires more care than normally used for smaller molecules. The general protocol used is the preparation of DNA directly in solid agarose blocks (plugs) or beads. Intact cells are encapsidated in agarose (plugs or beads) and are treated with different combinations of enzymes and detergent to remove cell walls, membranes, RNA, proteins, and other materials in order to obtain naked DNA. This is possible because detergent and enzymes can diffuse by Brownian motion through the agarose pores of the plug, whereas the large pieces of DNA cannot and remain sequestered inside the plug. Here, we give detailed protocols of preparation for yeast and mammalian DNA. In addition, we describe the power supplies, switchers, gels, and gel trays required for one-dimensional pulsed-field gel electrophoresis (ODPFGE), as well as the pulse strategies used for the separations.