Preparation of Chromosome-sized Yeast DNA Molecules in Solid Agarose1. 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 TE 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 TE with 1 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 42ºC.
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 C.
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 37ºC
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 50ºC
12. Wash plugs 4 time in pH 7.5 TE buffer ( 1 hour each wash).
13. Store at 4ºC in 0.05M EDTA, 0.01 M TRIS pH 7.5.
Program for Separating Chromosomes of Saccharomyces cerevisiaeSize range: 240-2200 kb
Agarose: 1.0% electrophoresis agarose
Buffer: 0.5x TBE
Temperature: 14ºC
Switch time: 60-120 seconds
Run time: 24 hours
Angle: 120º
Voltage gradient: 6V/cm
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