Skip to main content
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1988 Aug 11;16(15):7563–7582. doi: 10.1093/nar/16.15.7563

Optimized conditions for pulsed field gel electrophoretic separations of DNA.

B W Birren 1, E Lai 1, S M Clark 1, L Hood 1, M I Simon 1
PMCID: PMC338427  PMID: 3412895

Abstract

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. We have also varied the angle between the alternating electric fields. Reorientation angles between 105 degrees and 165 degrees 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.

Full text

PDF
7572

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Carle G. F., Frank M., Olson M. V. Electrophoretic separations of large DNA molecules by periodic inversion of the electric field. Science. 1986 Apr 4;232(4746):65–68. doi: 10.1126/science.3952500. [DOI] [PubMed] [Google Scholar]
  2. Carle G. F., Olson M. V. Orthogonal-field-alternation gel electrophoresis. Methods Enzymol. 1987;155:468–482. doi: 10.1016/0076-6879(87)55031-5. [DOI] [PubMed] [Google Scholar]
  3. Carle G. F., Olson M. V. Separation of chromosomal DNA molecules from yeast by orthogonal-field-alternation gel electrophoresis. Nucleic Acids Res. 1984 Jul 25;12(14):5647–5664. doi: 10.1093/nar/12.14.5647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chu G., Vollrath D., Davis R. W. Separation of large DNA molecules by contour-clamped homogeneous electric fields. Science. 1986 Dec 19;234(4783):1582–1585. doi: 10.1126/science.3538420. [DOI] [PubMed] [Google Scholar]
  5. Ellis T. H., Cleary W. G., Burcham K. W., Bowen B. A. Ramped field inversion gel electrophoresis: a cautionary note. Nucleic Acids Res. 1987 Jul 10;15(13):5489–5489. doi: 10.1093/nar/15.13.5489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Herrmann B. G., Barlow D. P., Lehrach H. A large inverted duplication allows homologous recombination between chromosomes heterozygous for the proximal t complex inversion. Cell. 1987 Mar 13;48(5):813–825. doi: 10.1016/0092-8674(87)90078-x. [DOI] [PubMed] [Google Scholar]
  7. Noolandi J, Rousseau J, Slater GW, Turmel C, Lalande M. Self-trapping and anomalous dispersion of DNA in electrophoresis. Phys Rev Lett. 1987 Jun 8;58(23):2428–2431. doi: 10.1103/PhysRevLett.58.2428. [DOI] [PubMed] [Google Scholar]
  8. Plasterk R. H., Simon M. I., Barbour A. G. Transposition of structural genes to an expression sequence on a linear plasmid causes antigenic variation in the bacterium Borrelia hermsii. Nature. 1985 Nov 21;318(6043):257–263. doi: 10.1038/318257a0. [DOI] [PubMed] [Google Scholar]
  9. Schwartz D. C., Cantor C. R. Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis. Cell. 1984 May;37(1):67–75. doi: 10.1016/0092-8674(84)90301-5. [DOI] [PubMed] [Google Scholar]
  10. Smith C. L., Matsumoto T., Niwa O., Klco S., Fan J. B., Yanagida M., Cantor C. R. An electrophoretic karyotype for Schizosaccharomyces pombe by pulsed field gel electrophoresis. Nucleic Acids Res. 1987 Jun 11;15(11):4481–4489. doi: 10.1093/nar/15.11.4481. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Snell R. G., Wilkins R. J. Separation of chromosomal DNA molecules from C.albicans by pulsed field gel electrophoresis. Nucleic Acids Res. 1986 Jun 11;14(11):4401–4406. doi: 10.1093/nar/14.11.4401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Southern E. M., Anand R., Brown W. R., Fletcher D. S. A model for the separation of large DNA molecules by crossed field gel electrophoresis. Nucleic Acids Res. 1987 Aug 11;15(15):5925–5943. doi: 10.1093/nar/15.15.5925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Vollrath D., Davis R. W. Resolution of DNA molecules greater than 5 megabases by contour-clamped homogeneous electric fields. Nucleic Acids Res. 1987 Oct 12;15(19):7865–7876. doi: 10.1093/nar/15.19.7865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Waterbury P. G., Lane M. J. Generation of lambda phage concatemers for use as pulsed field electrophoresis size markers. Nucleic Acids Res. 1987 May 11;15(9):3930–3930. doi: 10.1093/nar/15.9.3930. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES