Nucleic Acids Research, 1984, Vol. 12, No. 14 5647-5664
© 1984
Articles |
Separation of chromosomal DNA molecules from yeast by orthogonal-field-alternation gel electrophoresis
Department of Genetics, Box 8031, Washington University School of Medicine, St. Louis, MO 63110, USA
Received May 14, 1984. Accepted June 29, 1984.
A simple agarose-gel apparatus has been developed that allows the separation of DNA molecules in the size range from 50 kb to well over 750 kb, the largest size for which size standards were available. The apparatus is based on the recent discovery that large DNA molecules are readily fractionated on agarose gels if they are alternately subjected to two approximately orthogonal electric fields. The switching time, which was on the order of 20–50 sec in our experiments, can be adjusted to optinize fractionation in a given size range. The resolution of the technique is sufficient to allow the fractionation of a sanple of self-ligated 
DHA into a ladder of approximately 15 bands, spaced at 50 kb intervals. We have applied the technique to the fractionation of yeast DHA into 11 distinct bands, several of which have been shown by DNA-DNA hybridization to hybridize uniquely to different chromosome-specific hybridization probes. In this paper, we describe the design of the apparatus, the electrophoretic protocol, and the sample-handling procedures that we have employed.
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