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Nucleic Acids Research, 1988, Vol. 16, No. 15 7563-7582
© 1988

Articles

Optimized conditions for pulsed field gel electrophoretic separations of DNA

Bnice W. Birren, Eric Lai, Steven M. Clark, Leroy Hood and Melvin I. Simon

Division of Biology, California Institute of Technology Pasadena, CA 91125, USA

Received February 29, 1988. Revised June 28, 1988. Accepted June 28, 1988.

Quantitative measurement of DNA migration in gel electrophoresis requires precisely controlled homogeneous electric fields. A new electrophoresis system has allowed us to explore several parameters governing DNA migration during homogeneous field pulsed field gel (PFG) electrophoresis. Migration was measured at different switch times, temperatures, agarose concentrations, and voltage gradients. Conditions which increase DNA velocities permit separation over a wider size range, but reduce resolution. He have also varied the angle between the alternating electric fields. Reorientation angles between 105° and 165° give equivalent resolution, despite significant differences in DNA velocity. Separation of DNA fragments from 50 to greater than 7000 kilobases (Kb) can easily be optimized for speed and resolution based on conditions we describe.


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