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Nucleic Acids Research, 1994, Vol. 22, No. 24 5366-5370
© 1994

Articles

Rapid pulsed field separation of DNA molecules up to 250 kb

Bruce Birren*, and Eric Lai1

Division of Biology 147-75, California Institute of Technology Pasadena, CA 91125 1Department of Pharmacology, University of North Carolina at Chapel Hill Chapel Hill, NC 27599-7365, USA

*To whom correspondence should be addressed at: Whitehead Institute/MIT Center for Genome Research, 1 Kendall Square. Bldg 300. Cambridge. MA 02139, USA

Received August 9, 1994. Accepted October 14, 1994.

Pulsed field gel electrophoresis (PFGE) Is capable of resolving a wide size range of DNA molecules which would all co-migrate in conventional agarose gels. We describe pulsed field gel conditions which permit DNA fragments of up to 250 kllobases (kb) to be separated in only 3.5 h. The separations, which employ commercially available gel boxes, are achieved using conditions which deviate significantly from traditional pulsed field conditions. PFGE separations have been thought to require reorientation angles greater than 90° to be effective. However, reorientation angles of 90° and even less will resolve DNA fragments a few hundred kb and smaller {sum}5 x faster than with standard pulsed field conditions. The mobility of DNA fragments separated with 90° reorientation angles Is switch time-dependent, as is seen for DNA run with the commonly used reorientation angle of 120°. With DNA fragments of several hundred kb and smaller, higher field strengths may be used, resulting in still greater increases in separation speed. The conditions described allow DNA from large Insert bacterial clones, such as those using cosmld, Fosmld, P1, bacterial artificial chromosome (BAC), or P1-derived artificial chromosome (PAC) vectors, to be prepared, digested and analyzed on gels within a single working day.


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