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