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.
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
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
13. Meanwhile, add 150 ul of PFGE dye mixture to a gel slice containing
DNA that has been digested with restriction enzyme.
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
16. Visually verify that the agarose in the sample wells has solidified.
17. Complete assembly of the electrophoresis chamber, and resume
18. Turn on power supply, and set to regulate field strength at
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