Pulsed Field Gel Electrophoresis

Pulsed Field Gel Electrophoresis

Preparation of Chromosome-sized Yeast DNA Molecules in Solid Agarose

(Modified from Schwartz and Cantor, CELL 37:67 1984 )

1.Grow 5 ml yeast culture in YPD Broth to " stationary" (OD600 10-14)

2.Transfer 1ml to a microfuge tube ( PGC Scientific 2.2ml tube #509-220)

3.Pellet cells (20 seconds) and resuspend in 1ml pH 7.5 TE50 buffer ( 0.05 M EDTA, 0.01 M TRIS pH 7.5)

4.Wash twice with pH 7.5 TE buffer.

5.Resuspend in 0.15 ml of pH 7.5 TE50 with 2 µl zymolyase ( 20 mg/ml in 10 mM sodium phosphate pH 7.5)

6.Add 0.25 ml of 1% low melting agarose ( 1% low melting agarose in 0.125 M EDTA pH 7.5) at 42℃.

7.Immediately (<5 min) after mixing zymolyase and agar, gently mix with blue pipeete tip (3-5X) and put into CHEF disposable plug molds (Bio-Rad, 170-3713) which is cooled on ice. Zymolyase appears to become inactive upon prelonged incubation at 42 ℃.

8.Transfer plugs to a tube and add 0.4 ml LET ( 0.5 M EDTA, 0.01 M TRIS pH 7.5)

9.Incubate 8-10 hours or o/n at 37℃

10.Transfer plugs to a 12X 75 falcon tube containing 0.4 ml NDS, pH9.5 ( 0.5M EDTA, 0.01 M TRIS, 1% N-Lauroyl sarcosine, 2mg/ml proteinase K ). Add proteinase K fresh.

11.Incubate o/n at 50℃.

12.Wash plugs 4 time in pH 7.5 TE buffer ( 1 hour each wash).

13.Store at 4℃ in 0.05M EDTA, 0.01 M TRIS pH 7.5.

Program for Separating Chromosomes of Saccharomyces cerevisiae

Size range: 240-2200 kb

Agarose: 1.0% electrophoresis agarose

Buffer: 0.5x TBE

Temperature: 14℃

Switch time: 60-120 seconds

Run time: 24 hours

Angle: 120º

Voltage gradient: 6V/cm