PULSE FIELD GEL ELECTROPHORESIS (PFGE)

Preparation of Cells:
High molecular w eight DNA is needed for this procedure. Cells are lysed in agarose to avoid the shearing effects of pipeting. This method is a modification of G.T. Williams, Gene 53:121-128 (1987), and G.T. Williams et al, Nature 324:63 (1986).

  1. When using human peripheral blood leukocytes, eliminate as many of erythrocytes as possible with RBC NH4Cl Lysing Solution at 37°C for 5 minutes, followed by centrifugation of the leukocytes at 800 rpm for 5-6 minutes. Repeat as necessary. Lymphocytes may be purified by density gradient centrifugation or treatment with antibodies.
  2. Centrifuge the cell suspension for 5 minutes, 4°C, 800 rpm and transfer to ice. Remove the supernatant and resuspend the cell pellet in 1 ml of ice-cold Tris-buffered saline or PBS. Centrifuge again and resuspend in 1 ml of Tris-buffered saline or PBS. Keep on ice.
  3. Determine the cell concentration.
  4. Transfer the desired number of cells to microcentrifuge tubes. Optimal numbers of cells seem to be about 1.5 x 106. Centrifuge the cells as above. Remove the supernatant and transfer the pellet to ice. Centrifuge the pellet for 15 seconds in a microcentrifuge at 4°C. Remove the remaining supernatant with a Pipetman.
  5. Freeze the tubes immediately at -70°C for future use.
Preparation of DNA:
  1. Melt 2% l ow melting temperature agarose (FMC Sea Plaque) in buffer A at 65°C. Allow this solution to equilibrate to 50°C in a heat block.
  2. Weigh proteinase-K to bring to 0.5 - 1.0 mg/ml in the above agarose solution. Mix with agarose just before working with the cells in the next step.
  3. Remove cells from the -70°C freezer and allow them to thaw on ice. Centrifuge a few seconds in the microcentrifuge and remove any supernatant. Add 10 µl of ice-co ld buffer A and heat the cells to 50°C for 1 minute.
  4. Add 10 µl of the 2% agarose/proteinase-K solution to the cells. Mix very gently by swirling with a pipet tip (minimize pipeting!). Do not allow the sample to solidify (at about 30°C).
  5. Incubate at 50°C for 2 hours.
  6. Dialyze the detergent and the debris before restriction enzyme digestion by centrifuging the pellet for 3 seconds in the microcentrifuge. Cool on ice for 5 minutes to allow the agarose to solidify. Incubate with 500 µl of sterile TE at 4°C for 16 - 24 hours with 3 buffer changes.
  7. For restriction enzyme digests, remove the TE. Add 3.5 µl of 10x restriction enzyme buffer and H2O (for a 35 µl digest volume) to the pellet. Melt the samples at 65°C for about 5 minutes (sometimes this takes longer) and transfer to a 37°C heat block. Add 40-60 units of restriction enzyme. Mix quickly by stirring with a pipet tip so the agarose does not solidify. Incubate 18-24 hours at 37°C. Add more enzyme in the morning if needed. Depending on the enzyme's half-life, it may need to be added in small aliquots over time.
Sample Loading: DNA may be loaded as a molten agarose solution or as solidified agarose plugs.

For molten agarose solutions: QS the restriction enzyme digested samples with 1% low melting temperature agarose in 0.5x TBE to 65 µl. Mix gently by sw irling with a pipet tip and centrifuge the sample 10 seconds in the microcentrifuge. Melt the samples at 65-75°C and add 7.0 µl of stop dye. Continue to heat at 65-75°C for 10-15 minutes and pipet into the gel (see conditions below). Allow to solidify. Add molecular weight plugs. Top off all wells as needed with 1% low melting temperature agarose and allow to solidify.

To load the DNA as a solid agarose plug::
  1. Assemble the plug casting unit by clamping the solid plexiglass about 6 mm above the bottom of the wells (use clamps, not screws). Heat the casting unit in a 65°C oven for about 20 minutes.
  2. QS samples to 60 µl with 1% low melting temperature agarose in 0.5x TBE. Mix gently by swirling with a pipet tip and centrifuge the sample for 10 seconds in a microcentrifuge. Melt the samples at 65-75°C and add 6.0 µl of stop dye. Continue to heat at 65-75°C for 10-15 minutes and pipet into the pre-warmed casting stand. Allow to solidify at 4°C.
  3. Open the casting stand and remove plugs with small clean spatula. Place plugs on the side of the teeth of the PFGE comb that will face the running side of the gel. Incubate 37°C for no more than 5 minutes to adhere the plug to plastic. Mount in gel forming stand and carefully pour 150 ml of 0.8% PFGE certified agarose in 0.5x TBE. This solution should not be above 60°C or the plugs might melt. Allow to solidify.
  4. Remove comb leaving the plugs in the gel. Add molecular weight plugs. Fill wells with 0.8% low melting temperature agarose.
Gel Preparation (for use with the Bio-Rad CHEF/PFGE unit):
  1. Hook up the pump to circulate 0.5x TBE buffer through an ice bath and equilibrate the buffer to 14°C. Make about 2 L of buffer. Pump at about 1L per minute to avoid disturbing the gel.
  2. Prepare a 150 ml, 0.8% PFGE certified agarose gel in 0.5x TBE. The buffer should cover the gel to a depth of about 2-3 mm.
Electrophoresis:
Electrophoretic conditions will vary depending on the size of DNA you expect to examine. For a broad range of DNA from about 50kb to 2Mb we electrophorese at 200 constant volts 15 hours at 60 sec/60 sec switch times, and 8 hours at 90 sec/90 sec switch times. Stain with ethidium bromide for 20 minutes. Destain twice with H2O for 20 minutes each. Photograph.

Transfer Conditions:
Treat the gel in 300 ml of 0.25 M HCl for 15 minutes at room temperature. Rinse with a small amount of 0.5 M NaOH, 1.5 M NaCl denaturing solution. Then follow the Southern blot protocol for the actual transfer to a nylon membrane.

Recipes

RBC NH4Cl Lysing Solution
0.83 g NH4Cl
0.1 g KHCO3
90 µl of 10% EDTA
QS to 100 ml with H2O
Tris-buffered Saline
50 mM Tris-HCl, pH 8.0
150 mM NaCl
Buffer A
0.5% sodium N-lauroyl sarcosinate (helps solubilize proteins; Sigma L-5125)
10 mM EDTA
50 mM Tris-HCl, pH 8.0
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Copyright 1993, 1996, and 1997 by Mark Barton Frank, Ph.D.
Proper citation for data acquired from this document is: "Horowitz, R. and Frank, M. B. Pulse-Field Gele Electrophoresis. In: Frank, M. B. ed. Molecular Biol ogy Protocols. (http://omrf.ouhsc.edu/~frank/pfge.html). 1997. Oklahoma City. Revision Date: October 2, 1997."