7 - Orthogonal-Field-Alternation Gel Electrophoresis

Abstract

This chapter focuses on the detailed protocols for separating large DNA molecules by orthogonal-field-alternation gel electrophoresis (OFAGE). As the size of DNA molecules increases, it becomes progressively more difficult to keep them intact. To handle intact DNA molecules larger than 500 kb, it has proved necessary to prepare DNA samples by in situ lysis of cells or spheroplasts in a semisolid matrix. OFAGE results are influenced by an unusual number of interdependent variables. The functionally critical feature of an OFAGE apparatus is the positioning of the electrodes in relation to the gel and the perimeter of the electrophoresis chamber. OFAGE involves an element of brinksmanship in which the standard electrophoresis conditions border closely on disaster. It is probable that this situation is a direct reflection of the underlying fractionation principle, which is largely based on the retardation of large DNA molecules by interactions with the gel matrix that are only barely reversible.