Abstract
The preparation and manipulation of very large DNA molecules for pulsed-field gel electrophoresis (PFGE) requires more care than normally used for smaller molecules. The general protocol used is the preparation of DNA directly in solid agarose blocks (plugs) or beads. Intact cells are encapsidated in agarose (plugs or beads) and are treated with different combinations of enzymes and detergent to remove cell walls, membranes, RNA, proteins, and other materials in order to obtain naked DNA. This is possible because detergent and enzymes can diffuse by Brownian motion through the agarose pores of the plug, whereas the large pieces of DNA cannot and remain sequestered inside the plug. Here, we give detailed protocols of preparation for yeast and mammalian DNA. In addition, we describe the power supplies, switchers, gels, and gel trays required for one-dimensional pulsed-field gel electrophoresis (ODPFGE), as well as the pulse strategies used for the separations.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Lalande, M., Kunkel, L. M., Fhnt, A., and Latt, S. A. (1984) Development and use of metaphase chromosome flow sorting methodology to obtain recombinant phage libranes enriched for parts of the human X chromosome. Cytometry 5, 101–107.
Fem berg, A. P. and Vogelstem, B. (1983) A technique for radio-labelling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132, 6–13.
Turmel, C., Brassard, E., Forsyth, R., Hood, K., Slater, C. W., and Noolandi, J. (1990) High resolution zero integrated field electrophoresis (ZIFE) of DNA, in Current Communication in Molecular Biology. Electrophoresis of Large DNA Molecules (Birren, B. and Lal, E., eds.), Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 101–131.
Lalande, M., Noolandi, J., Turmel, C., Rousseau, J., and Slater, G. W. (1987) Pulsed-field electrophoresrs: Application of a computer model to the separahon of large DNA molecules. Proc. Natl. Acad. Sci. USA 84, 8011–8015.
Slater, G. W., Rousseau, J., Noolandr, J., Tunnel, C., and Lalande, J. (1988) Quantative analysis of the three regimes of DNA electrophoresis in agarose gels. Biopolymers 27, 509–524.
Tunnel, C., Brassard, E., Slater, G. W., and Noolandi, J. (1989) Molecular detrapping and band narrowing with high frequency modulation of pulsed field electrophoresis. Nucleic Acids Res. 18, 569–575.
Olson, M. V. (1989) Pulsed field gel electrophoresis, in Genetic Engineering (Setlow, J. K., ed.), Plenum, New York, pp. 183–227.
Heller, C. and Pohl, M. (1990) Freld inversion electrophoresis with different pulse time ramps. Nucleic Acids Res. 18, 6299–6304.
Buren, B. W., Lal, E., Clark, S. M. Hood, L., and Simon, M. I. (1988) Optimized conditions for pulsed field gel electrophoretic separations of DNA. Nucleic Acids Res. 16, 7563–7582.
Fan, J, B., Chikashige, Y., Smith, C. L., Niwa, O., Yanagida, M., and Cantor, C. R. (1988) Construction of a Not I restriction map of the fission yeast Schizosaccharomyces pombe genome. Nucleic Acids Res. 17, 2801–2818.
Lasker, B. A., Carle, G. F., Kobayashi, G. S., and Medoff, G. (1989) Comparison of the separation of Candida albicans chromosome-sized DNA by pulsed field gel electrophoresis techniques. Nucleic Acids Res. 17, 3783–3793.
Chu, G. (1990) Pulsed field gel electrophoresis: Theory and practice. Methods: A Companion to Methods Enzymol. 1, 129–142.
Higgins, M. J., Turmel, C., Noolandi, J., Neumann, P., and Lalande, M. (1990) Construction of the physical map for three loci in chromosome band 13q14: Comparison to the genetic map. Proc. Natl. Acad. Sci. USA 87, 3415–3419.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1992 The Humana Press Inc.
About this protocol
Cite this protocol
Noolandi, J., Turmel, C. (1992). Preparation, Manipulation, and Pulse Strategy for One-Dimensional Pulsed-Field Gel Electrophoresis (ODPFGE). In: Burmeister, M., Ulanovsky, L. (eds) Pulsed-Field Gel Electrophoresis. Methods in Molecular Biology™, vol 12. Humana Press. https://doi.org/10.1385/0-89603-229-9:73
Download citation
DOI: https://doi.org/10.1385/0-89603-229-9:73
Publisher Name: Humana Press
Print ISBN: 978-0-89603-229-3
Online ISBN: 978-1-59259-499-3
eBook Packages: Springer Protocols