Rapid pulsed field separation of DNA molecules up to 250 kb

Abstract

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 kilobases (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 degrees to be effective. However, reorientation angles of 90 degrees and even less will resolve DNA fragments a few hundred kb and smaller approximately 5 x faster than with standard pulsed field conditions. The mobility of DNA fragments separated with 90 degrees reorientation angles is switch time-dependent, as is seen for DNA run with the commonly used reorientation angle of 120 degrees. 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 cosmid, Fosmid, 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.

Inhibition of bacterial DNA replication by zinc mobilization during nitrosative stress

“…Protocol and buffers were adapted from the methods of Birren and Lai (15,16). Bacteria were grown anaerobically overnight in M9 with 0.2% glucose by using a BBL GasPak (Becton Dickinson).…”

Caenorhabditis elegans cog-1 Locus Encodes GTX/Nkx6.1 Homeodomain Proteins and Regulates Multiple Aspects of Reproductive System Development

“…DNA sequences were analyzed with the GCG software package (Devereux et al, 1984) and the Mac Vector program (IBI). YAC DNA was isolated as described (Birren and Lai, 1994) and was injected at a concentration ranging from 10 to 20 g/ml. Cosmid and plasmid clones were injected at concentrations of ϳ25 g/ml.…”

Genetic and Physical Mapping of the Dreher Locus on Mouse Chromosome 1

“…Digested DNA was run for 3.5 h in a 0.8% agarose gel in 0.5X TAE buffer on a CHEF DRII apparatus (Bio-Rad Laboratories, Hercules, CA). Reorientation angles were 100°with a field strength of 9 V/cm and a switch interval that was linearly ramped from 3 to 5 s (Birren and Lai, 1994).…”