Yeast transformation

General:

  • Carry out 1 transformation with DNA and 1 without DNA (no-DNA control is used to count the number of revertants)

Materials and equipment needed:

  • Fresh stationary-phase yeast
    • 5 ml suffices for both 1 transformation and 1 control
  • Linearised DNA (either PCR product or linearized plasmid)
    • ~1 ug for 1 transformation
  • Drop-out plates (-Leu, -His, -Trp, -Ura, +G418, or +ClonNat)

  • 1x Lithium acetate-TE solution (LiOAc/TE)
    • Need: 200 ul suffices for both 1 transformation and 1 control
    • 5x LiOAc/TE stock:
      • 0.5 M LiOAc (51 g of 99% LiOAc•2H20 per 1 l)
      • 5x TE (50 mM Tris and 5 mM EDTA)
  • 1x PEG-LiOAc/TE solution (make fresh)
    • Need: 2 ml suffices for both 1 transformation and 1 control
    • 40% PEG (we and others use: 3350 MW) in 1x LiOAc/TE
    • Example: to make 2.5 ml, mix 0.5 ml of 5x LiOAc/TE with 2 ml 50% PEG solution
  • 1x Carrier DNA
    • Need: 60 ul suffices for both 1 transformation and 1 control
    • 10 mg/ml Herring sperm
  • Water bath at 42 C
  • Optional: pasteur pipettes for plating

Procedure:

  1. Pellet yeast cells at ~700 g for 2 min
  2. Check for bacterial contamination (supernatant should not be cloudy)
  3. Pour supernatant off carefully
  4. Re-suspend yeast cells in 200 ul of 1x LiOAc/TE
  5. Label 1.5 ml microcentrifuge tubes for transformations and control
  6. Heat carrier DNA to 70 C for 5 min
  7. Add 30 ul of carrier DNA to microcentrifuge tubes
  8. Add ~1 ug of DNA for transformation, no DNA for control
  9. Add 100 ┬Ál of yeast cells to microcentrifuge tubes (with and without DNA)
  10. Vortex
  11. Add 1 ml of 1X PEG-LiOAc/TE solution to each tube
  12. Vortex
  13. Incubate at 42 C for 30 min
  14. Pellet at ~700 g for 2 min
  15. Resuspend in 1 ml DI water
  16. Pellet at ~700 g for 2 min
  17. Plate or let grow overnight and then plate:
    • For all selectable markers (LEU2, HIS3, TRP1, URA3) except KanMX and NatMX:
      • Add 200 ul DI water
      • Pipette up and down to mix
      • Streak onto dropout plates
    • For KanMX and NatMX plasmids:
      • Add 1 ml non-selective medium (e.g., D-Met)
      • Important: Lock tubes shut (often tubes pop open)
      • Put on nutator overnight at 30 C
      • Pellet at ~700 g for 2 min
      • Add 200 ul DI water
      • Pipette up and down to mix
      • Streak onto G418 or ClonNAT plates

Author: Enrico Tenaglia
September 2018