Molecular Techniques and Methods

Pulsed Field Gel Electrophoresis (PFGE)
for Genomic DNA Fingerprinting

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

The specialized electrophoretic methods required to separate populations of large DNA fragments into countable numbers of gel bands are collectively called pulsed-field gel electrophoresis (PFGE). During standard, single orientation electrophoresis, the varying molecular sieving effects of the gel matrix on DNA molecules of different sizes leads to their separation. However, above a certain size, differently sized DNA molecules tend to migrate at indistinguishable rates in a unidirectional field, probably because of molecular tunneling through the porous gel matrix and the consequent loss of the sieving effect. PFGE techniques depend on periodic switching of the orientation of the electric field during electrophoresis. The size-dependent rate of reorientation of DNA molecules within this variable electric field causes the molecular species to be separable over a much wider size range. Varying the parameters of the electric field (e.g., voltage/ time interval between successive reorientations/ angle or reorientation) allows optimization of the size range of DNA separated.




MATERIALS AND SOLUTIONS

Agarose (Multipurpose)
Boehringer-Mannheim MP agarose has high gel strength and yields gels of excellent quality.


5 x TBE Buffer (1 liter)
Boric acid --------------------------------- 27.5 g
Trizma base -------------------------------- 54 g
EDTA-Na2 -------------------------------- 1.86 g
Add distilled H2O to make a final volume of ----- 1 liter
  • Adjust pH to 8.6.




    PROCEDURES

    1. Dissolve pulsed-field grade agarose in 1 x TBE electrophoresis buffer to a final concentration of 1 %.

    2. Heat in a microwave oven for 3 minutes.

    3. Swirl the solution gently to release trapped air and resuspend any agarose particles caught on the side of the flask.

    4. Heat in a microwave oven for additional 1 minutes.

    5. Cool down the agarose solution to 50-55oC.

    6. Pour the 1% agarose solution smoothly into the gel casting tray.

    7. Once the gel has solidified, place at 4oC for 30 minutes to complete gelation.

    8. Set the thermostatic circulator of the electrophoresis apparatus to a temperature that will maintain a buffer temperature of 14oC in the electrophoresis chamber.

    9. Place the 1 x TBE electrophoresis buffer into the electrophoresis chamber and level the apparatus.

    10. Slowly remove the well-forming comb from the gel.

    11. Place gel on the casting tray, on the platform in the chamber.

    12. Begin circulating the electrophoresis buffer to bring it to 14oC.

    13. Meanwhile, add 150 ul of PFGE dye mixture to a gel slice containing DNA that has been digested with restriction enzyme.
  • Incubate the slice at 65oC for 15 minutes until the agarose has completely melted.

    14. Stop circulation of the electrophoresis buffer.

    15. Take one sample out of the 65oC water bath and promptly load 20-25 ul of sample mixture in a sample well.

    16. Visually verify that the agarose in the sample wells has solidified.

    17. Complete assembly of the electrophoresis chamber, and resume buffer circulation.

    18. Turn on power supply, and set to regulate field strength at 200 volts.
  • Adjust pulse-ramping interval to an empirically determined optimal value (1-30 seconds over the course of the run) for the size range of DNA fragments expected.

    19. Apply power and run the electrophoresis for 18-24 hours, or until the bromophenol blue marker has migrated 10-15 cm from the sample wells.

    20. Turn off power supply.

    21. Carefully lift the gel out of the chamber on the supporting gel tray, and place into 500 ml 0.5-1 ug/ ml ethidium bromide stain solution.

    22. Agitate gently for 30-60 minutes.

    23. Destain the gel with 500 ml water for 30-60 minutes.

    24. Record and analyze banding patterns on the ultraviolet transilluminator at 254 nm (for highest sensitivity) or 310 nm (for less sample DNA damage and for band recovery).


    NOTES



    KIT INFORMATION



    REFERENCES


  • Please send your comment on this protocol to "editor@MolecularInfo.com".

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