Yeast DNA plugs for PFGE were prepared according to the protocol in the Genome Analysis Laboratory Handbook (Birren, et al 1997).

Harvest 2.5x10^8 diploid cells, or twice as many haploids. Resuspend in 20 ml 50 mM EDTA pH 8.
Spin and discard supernatant.
Resuspend in 2 ml SCE solution.
Spin and discard supernatant. Drain residual.
Add 150 microliters yeast plug and bead spheroplasting solution.
Resuspend with gentle pipetting.
Incubate 37 degrees C 15 minutes.
While incubating, prepare a 2% low melting temperature, CHEF-quality, agarose solution in 50 mM EDTA (pH 8) and equilibrate to 45 degrees C.
Add 250 microliters of agarose to cell suspension and mix gently but thoroughly by pipetting up and down a few times.
Prepare the molds beforehand by taping up the holes.
Fill molds with cell suspension.
Ice or place at 4 degrees C for 10 minutes to solidify.
Remove tape and slide plugs into 10X volume of plugs of EDTA/Tris.mercaptoethanol buffer. Seal up to contain fumes.
Incubate 37 degrees C overnight with or without agitation.
Aspirate solution. Add 10X volume of proteinase K solution for preparing HMW yeast DNA. Seal up.
Incubate 50 degrees C overnight without agitation.
Aspirate. Add 10X volume 50 mM EDTA (pH 8).
Agitate for 30 minutes at room temperature.
Store in fresh 50 mM EDTA at 4 degrees C.

SCE solution (pH 7)
1 M sorbitol
100 mM sodium citrate dihydrate trisodium salt
10 mM EDTA

Dissolve in 0.8 L water and allow to come to room temperature. pH to 7.0 with hydrochloric acid. Adjust volume to 1 L. Sterilize. Store room temperature indefinitely.

Yeast plug and bead spheroplasting solution
1 M sorbitol
20 mM EDTA
10 mM Tris-Cl
14 mM beta-mercaptoethanol
1 mg/ml yeast lytic enzyme (70 U/mg) or same units of lyticase

Dissolve sorbitol in water to final volume of 0.9 L. Allow to come to room temperature. Add EDTA and Tris. pH to 7.5 with hydrochloric acid. Sterilize with 0.22 micron filter. Store room temperature indefinitely. Just before use, add BME and lyticase.

EDTA/Tris/mercaptoethanol buffer
0.5 M EDTA
10 mM Tris-Cl
1 part beta-mercaptoethanol:250 parts buffer

Prepare just before use

Proteinase K solution for preparing HMW yeast DNA
10 mM Tris-Cl
0.5 M EDTA
1% (w/v) N-lauroylsarcosine sodium salt
0.5 mg/ml proteinase K

Combine Tris-Cl, EDTA, and sodium N-lauroylsarcosine. Filter sterilize. Store room temperature indefinitely. Just before use, add proteinase K.

Running Gel

Pour a 1% CHEF-quality agarose gel in 0.5X TBE.
While gel hardens, fill CHEF apparatus with 0.5X TBE and allow to chill.
Load plugs in wells, avoiding air bubble. I find a spatula works well for this.
Drip some cooled molten agarose on the wells to seal.
Put gel tray in apparatus.
Run gel at 6 V/cm for 15 hours at a switch time of 70 second. Run another 11 hours at a switch time of 120 seconds.
Stain gel in 0.5 micrograms/ml ethidium bromide for 30 minutes at 4 degrees C.
Photograph.