1. Streak yeast cell EGY48 with p8op-lacZ reporter plasmid on SD-Ura plate for 3-5 days (at 30 ^oC). Scrape an entire colony (2-3 mm in diameter) into 1 ml of YPD medium, Vortex vigorously to disperse any clumps. Transfer this into a flask containing YPD:
Small Scale: 50 ml in 250 ml flask
Library Scale: 150 ml in 500 ml flask

Incubate at 30 ^oC overnight (16-18 hr) with shaking (at 250 rpm) to stationary phase (OD₆₀₀ greater than/equal to 1.5).

2. Early the next morning, transfer overnight culture (enough to produce an OD₆₀₀=0.2-0.3) into this volume of YPD:
Small Scale: 300 ml in 1L flask
Library Scale: 1000 ml in 2L flask

3. Incubate at 30 ^oC for 3 hr with shaking (230 rpm). The OD₆₀₀ will be 0.5 plus/minus 0.1.

4. Place cells in 50-ml tubes and centrifuge at 1500 rpm in floor centrifuge for 5 min at room temperature.

5. Discard the supernatant and resuspend cell pellets by vortexing in this volume of sterile TE or H₂O:
Small Scale: 25-50 ml
Library Scale: 500 ml

6. Pool cells in 50 ml tube and centrifuge at 1500 rpm in floor centrifuge for 5 min at room temperature.

7. Decant the supernatant.

8. Resuspend the cell pellet in this volume of freshly prepared, sterile 1xTE/LiAc:
Small Scale: 1.5 ml
Library Scale: 8 ml

(NOTE: Cells must be used within about an hour after preparation.)

9. Prepare PEG/LiAc solution, and this solution will be used in step #12:
Small Scale: 10ml
Library Scale: 100ml

10. Add the following to each tube and mix:
Small scale (in 1.5 ml eppt tube)
Library scale (in 500 ml centrifuge tube)

Bait vector- 0.2 ug (small), 0.6 mg (library)
Library vector- 0.1 ug (small), 0.3 mg (library)
Herring testes carrier DNA- 0.1 mg (small), 20 mg (library)

(For simultaneous cotransfection, a molar ratio of 2:1 (Bait vector: Prey vector) is recommended). (Just prior to use, denature the carrier DNA by placing it in a boiling water for 20 min and immediately cooling it on ice)

11. Add this volume of yeast competent cells to each tube and mix well by vortexing:
Small Scale: 0.1 ml
Library Scale: 8 ml

12. Add sterile PEG/LiAc solution (made in step #9) to each tube and vortex at high speed to mix:
Small Scale: 0.6 ml
Library Scale: 60 ml

13. Incubate at 30 ^oC for 30 min with shaking (200 rpm).

14. Add this volume of DMSO:
Small Scale: 70 ul
Library Scale: 7 ml

Mix well by gentle inversion or swirling. Do not Vortex.

15. Heat shock for 15 min in a 42 ^oC water bath. Swirl occasionally to mix (large- and library-scale only)

16. Chill cells on ice for 1-2 min.

17. Centrifuge cells for:
Small Scale: 5 sec., room temperature - 14K rpm
Library Scale: 5 min., room temperature - 1500 rpm

18. Remove the supernatant.

19. Resuspend cells in this volume of 1 x TE:
Small Scale: 0.5 ml
Library Scale: 10 ml

20. Plating:
For Small-scale cotransformation: spread 200 ul per 100-mm SD-Ura-His-Trp.
For large- and library-scale transformation:
--- Spread 100 ul of a 1:1,000, 1:100, and 1:10 dilution on SD-Ura-His-Trp plates (100 mm) for cotransformation efficiency controls.
--- Spread 1 ul (diluted in 100 ul of H₂O) on SD-His-Ura and SD-Trp-Ura plates (100 mm) to check the transformation efficiency of each plasmid.
--- Spread the remaining transformation suspension on SD-Ura-His-Trp plates (200 ul per 150-mm Plates)
Incubate the plates at 30 ^oC for 3-5 days.

21. Calculating cotransformation efficiency and number of clones amplified:

A. Cotransformation efficiency: count the colonies (cfu) growing on the SD-His-Trp-Ura dilution plate that has 30-300 cfu.

cfu/ug DNA = (Cfu x total suspension vol. (ul)) /
((Vol. Plated (ul)) x (dilution factor) x (amount.DNA used(ug)(Limiting plasmid)))

B. The number of clones amplified:
cfu/ug x amt. Of library plasmid used (cfu/ug) = # of clones amplified.

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