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        脉冲凝胶电泳

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        2272
        Preparation of Chromosome-sized Yeast DNA Molecules in Solid Agarose

        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 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 cerevisiae

        Size 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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