Yeast DNA Prep protocol

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Solutions/reagents:

Equipment:

  • Centrifuge
  • Eppendorf tubes

Steps:

  1. Measure out 1.5 ml of yeast culture grown up to appropriate density into Eppendorf tube (1).
    Centrifuge at maximum speed for 1 min at room temperature, gently aspirate out the supernatant and discard it.
  2. Measure out 200 µl of breaking buffer into Eppendorf tube (1).
    Resuspend pellet by vortexing/by shaking vigorously.
  3. Add 200 µl of ice-cold phenol : chloroform : isoamyl alcohol.
    Add 200 mg of glass beads.
    Wear gloves for this step!
  4. Vortex the mixture for 2.5 mins .
    Be careful when vortexing; label can be dissolved by the phenol.
    Hold cap tightly so it doesn't open or spill.
  5. Add 200 µl of TE buffer.
    Centrifuge at maximum speed for 5 mins at room temperature and aspirate out 350 µl of top layer.
    Transfer top aqueous layer into Eppendorf tube (2).
    Discard bottom layer.
  6. Measure out 1 ml of ice-cold 95% ethanol into Eppendorf tube (2).
    Vortex the mixture for a few secs.
    Store at room temperature for 10 mins.
  7. Centrifuge at maximum speed for 2 mins at room temperature, gently aspirate out the supernatant and discard it.
    Stand the tube containing pellet for 10 mins in an inverted position on a paper towel to allow all of the fluid to drain away.
  8. Option 1: Add 50 µl of TE buffer.
    (or)
    Option 2: Add 50 µl of water.

    Resuspend pellet by vortexing/by shaking vigorously.
    You can now use 1-2 ul of this crude yeast plasmid DNA prep to transform E. coli.

TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 30 mins

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