High Efficiency Yeast Transformation

Modified from Gietz (2002) by Greg Foley and then Rishi Jajoo

MATERIALS

  • Filter-sterilized, 1M lithium acetate.
  • Filter-sterilized, 100mM lithium acetate.
  • Polyethylene glycol (MW = 3015-3685) 50% w/v. (10 g in 20 ml total volume)
  • Antibiotic YPD agar plates.
  • 2 ng/ ul Salmon Sperm

PROTOCOL

Day 1:

  1. 5:00 PM: Inoculate the yeast strain into 5 ml of YPD and incubate in shaker overnight.
  2. Place 50 ml of YPD in shaker. (This is enough for 10 Reactions)

Day 2

  1. Pipette 500 ul of overnight culture into 50 ml of pre-warmed YPD.
  2. Incubate in shaker for 4 hours.
  3. Boil a 1.0 ml sample of salmon sperm for 5 min and place on ice while harvesting the cells.
  4. Harvest the cells by centrifugation at 3000 g for 5 min in a 50 ml Falcon tube.
  5. Re-suspend cells in 25 ml of sterile water.
  6. Centrifuge at 3000 g for 5 min.
  7. Re-suspend in 1 ml of 100 mM lithium acetate.
  8. Transfer the cell suspension to a 1.5 ml microcentrifuge tube.
  9. Centrifuge for 30 seconds at max speed.
  10. Resuspend in enough 100 mM lithium acetate to reach 500 ul total volume.
  11. Make one, 50 ul aliquot for each transformation.
  12. Centrifuge the aliquots at top speed for 30 seconds and pipette off the supernatant.
  13. Add the following reagents to each transformation in the order given:
    • Polyethylene glycol 3500 50% w/v: 240 µl
    • Lithium acetate 1.0 M: 36 µl
    • Boiled salmon sperm (
    • Plasmid DNA plus Water (use 0.1 – 10
  14. Vortex.
  15. Incubate for 30 minutes at 30 degrees.
  16. Incubate at 42 degrees for 20 minutes.
  17. Centrifuge at top speed for 30 sec and pipette off the supernatant.
  18. Pipette 1.0 ml of YPD into each tube;
  19. Let sit 10 minutes to soften the pellet.
  20. Stir the pellet gently with a pipette tip and vortex for 1-2 seconds until the cells are re-suspended.
  21. Burn a hole through the top of the epitube with a hot 20 gauge needle to prevent fermentation induced blowouts.
  22. Incubate at 30°C overnight.
  23. Plate 200ul aliquots onto selective plates.

Incubate the plates at 30°C for 3 to 4 days.