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Library-scale transformation of yeast

(Longo)

Purpose: To introduce library plasmid DNA into bait-expressing yeast.

Materials:
  • SD/Trp- medium
  • 10x TE, pH 7.5
  • 10x Lithium acetate

Procedure:
  1. Inoculate 2 ml of SD/Trp- medium with 10 colonies of yeast strain CG1945 expressing CR2#3 (clone "I"). Vortex vigorously, and use all 2 mL to inoculated 100 mL of SD/Trp- medium with 2% glucose in a 1 L flask.
  2. Cells are incubated overnight at 30 °C and 250 rpm.
  3. 2x50 mL aliquots of the overnight culture are used to inoculate 2x300 mL of SD/Trp-/+glucose. The cells are incubated at 30°C and 250 rpm for 5 hrs.
  4. The cells are harvested in 250 mL bottles and centrifuge (1000 xg, 5 min)
  5. The pellets are resuspended in a total of 50 mL of sterile 1x TE, pH 7.5, and pool
  6. The cells are centrifuged in one tube (1000 xg, 5 min).
  7. Decant supernatant.
  8. Resuspend pellet in 1 mL of 1X TE/LiAc.
  9. Prepare 10 mL of PEG LiAc solution.
  10. Add the following to one 50 mL conical centrifuge tube: 
    • 50 mg library DNA
    • 2 mg Herring testes carrier DNA
    • 1 mL of yeast competent cells
  11. Vortex vigorously
  12. Add 6 mL sterile PEG/LiAc solution and vortex vigorously.
  13. Incubate at 30°C for 30 min at 250 rpm.
  14. Prepare 42°C waterbath.
  15. Add 700 mL of DMSO to the cells. Mix gently by inversion and swirling. DO NOT VORTEX.
  16. Heat shock cells for 15 min at 42°C. Swirl occasionally to mix
  17. Chill cells on ice for 2 min.
  18. Remove supernatant.
  19. Resuspend the cell pellet in 4 mL of 1X TE
  20. Plate 100 mL of yeast cells per 150 mm Trp-/Leu-/His- plate.
  21. Make the following serial dilutions of the remaining yeast, and plate 100 mL per dilution on SD/Trp-/Leu- plates: 1:10, 1:100, 1:1000
  22. Place plates in 30°C incubator for 3-5 days until colonies form