EMS Mutagenesis

  1. Grow up a single colony in 100 ml YPD to stationary phase, O.D.(600) = 10.

  2. Spin down two separate 50 ml aliquots of the culture.

  3. Wash the pellet twice in 25 ml sterile 50 mM potassium phosphate buffer, pH 7.0.

  4. Resuspend both washed cell pellets in 4 ml 50 mM potassium phosphate buffer and combine into one tube.

  5. Cell concentration should be greater than 30 O.D.(600).

  6. Add 3 ml 50 mM potassium phosphate buffer to a 15 ml conical tube.

  7. Add 300 ul EMS, tighten cap, and vortex thoroughly.

  8. Add 7 ml of cells to tube and vortex. Start timer upon addition of cells.

  9. Aliquout 1 ml of cell suspension into 2 ml tubes.

  10. Aliquot 1 ml of untreated cells to a 2 ml microfuge tube and incubate as control for 90 minutes.

  11. Incubate at 30oC. Withdraw 1 ml aliquots at 15, 30, 45, 60 and 90 minutes.

  12. Add 1 ml of a fresh, filtered solution of 10% (w/v) sodium thiosulfate to each tube immediately upon removal from the original reaction.

  13. Spin down cells for 2 minutes at 600 rpm in microcentrifuge.

  14. Transfer to 1.7 ml tube and wash three times with sterile water. Resuspend in 1 ml sterile water. Take 10 ul to measure O.D.(600).

  15. Store remainder of treatment at 4oC.

    ***BEWARE OF EMS! WEAR A LAB COAT, DOUBLE GLOVES AND PERFORM ALL OPERATIONS IN THE FUME HOOD. RESERVE A BAG FOR ALL EMS-TAINTED SOLIDS.