Pulsed‐Field Gel Electrophoresis

Michael Finney1

1 MJ Research, Watertown, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 2.5B
DOI:  10.1002/0471142727.mb0205bs51
Online Posting Date:  May, 2001
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Abstract

DNA molecules longer than 25 kb are poorly resolved by standard agarose gel electrophoresis. These longer molecules can be resolved using several techniques that periodically change the direction of the electric field in the gel. This unit describes the simplest and most generally useful of the pulsed‐field techniques, field inversion electrophoresis, which can be tuned to resolve molecules from ˜10 to 2000 kb (or more with specialized equipment). To resolve molecules beyond the range of field inversion, it is necessary to use some sort of field‐angle alternation electrophoresis such as CHEF (contour‐clamped homogeneous electric field; described in an ). A method is also provided for preparing high‐molecular‐weight DNA samples and size markers embedded in agarose blocks.

     
 
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Table of Contents

  • Basic Protocol 1: Field‐Inversion Electrophoresis
  • Alternate Protocol 1: CHEF Electrophoresis
  • Support Protocol 1: Preparation of High‐Molecular‐Weight DNA Samples and Size Markers
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Field‐Inversion Electrophoresis

  Materials
  • 1% agarose gel, standard or pulsed‐field grade (e.g., SeaKem FastLane; FMC Bioproducts)
  • recipeGTBE buffer (see recipe) or 0.5× TBE buffer ( appendix 22)
  • protocol 3Samples embedded in agarose ( protocol 3), or liquid samples
  • Peristaltic pump (Cole‐Parmer Masterflex or equivalent)
  • Programmable switching device (MJ Research PPI‐200 or equivalent)
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.5)
NOTE: Some power supplies have pulsed‐DC rather than constant‐voltage output and are unacceptable for pulsed‐field gels. These can usually be recognized because their output is fixed, or adjustable in steps, rather than continuously variable.

Alternate Protocol 1: CHEF Electrophoresis

  • CHEF electrophoresis voltage‐divider circuitry and gel box (see ).

Support Protocol 1: Preparation of High‐Molecular‐Weight DNA Samples and Size Markers

  Materials
  • 1% agarose
  • Sample to be prepared (e.g., tissue culture cells, nematode worms, nuclei, yeast, bacteria, or phage; Table 2.5.1)
  • recipeLysis buffer (see recipe)
  • recipeStorage buffer (see recipe)
  • 400 mM phenylmethylsulfonyl fluoride (PMSF) in ethanol
  • 10 mM Tris·Cl, pH 8.0 ( appendix 22)
  • Appropriate restriction enzyme and buffer (unit 3.1)
  • Block molds or petri plates
    Table 2.5.1   MaterialsPreparation of High‐Molecular‐Weight DNA Samples and Size Markers

    Starting material Preparation
    Bacteria and phage Resuspend bacteria (unit 1.2) or phage particles (unit 1.13) at a concentration calculated to yield the desired amount of DNA per lane; e.g., 5 × 108E. coli per ml will yield ∼100 ng DNA in an average lane.
    Lambda ladders for size markers Start with a concentrated stock of phage λ particles (unit 1.13). This procedure does not work well with some lots of commercial λ DNA, possibly because of damaged cohesive ends. Try several dilutions of phage stock to see which works best. The second incubation in lysis buffer should be done at 25° rather than 37°C; during this incubation, the cohesive ends of the molecules will anneal, giving multimers of varying lengths. These concatemers will stay together during electrophoresis provided that the gel is run at <25°C.
    Nematodes Anesthetize worms by resuspending in 10 mM NaN 3, then place in agarose.
    Nuclei Isolate as in unit 4.10. Approximately 1 µg DNA is contained in 105 mammalian nuclei.
    Tissue culture cells Cells should be washed several times in a medium containing no serum, as serum may inhibit proteinase K.
    Yeast Saccharomyces cerevisiae cells must have their cell walls removed before being embedded in agarose as described in unit 13.13, protocol 1.

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Figures

Videos

Literature Cited

Literature Cited
   Birren, B.W., Lai, E., Clark, S.M., Hood, L., and Simon, M.I. 1988. Optimized conditions for pulsed field electrophoretic separations of DNA. Nucl. Acids Res. 16:7563‐7581.
   Carle, G.F., Frank, M., and Olson, M.V. 1986. Electrophoretic separations of large DNA molecules by periodic inversion of the electric field. Science 232:65‐68.
   Chu, G. 1989. Pulsed field electrophoresis in contour‐clamped homogeneous electric fields for the resolution of DNA by size or topology. Electrophoresis 10:290‐295.
   Chu, G., Vollrath, D., and Davis, R.W. 1986. Separation of large DNA molecules by contour clamped homogeneous electric fields. Science 234:1582‐1585.
   Schwartz, D.C. and Cantor, C.R. 1984. Separation of yeast chromosome‐sized DNAs by pulsed‐field gradient electrophoresis. Cell 37:67‐75.
Key Reference
   Schwartz and Cantor, 1984. See above.
  Describes the original use of pulsed fields to separate large molecules.
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