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1. Pick a single yeast colony from the plate and incubate in 10 ml YEPD at 30¡ C overnight with
shaking.
2. Add 1 ml of the overnight culture into 50 ml YEPD and incubate at 30¡ C overnight, OD»1.5.
3. Transfer about 30 - 40 ml of this culture to 300 ml YEPD to produce an OD=0.2. Incubate at 30¡ C with shaking for 3 - 4 hours.
4. Pellet the cells in a GSA rotor at 5000 rpm for 5 min.
5. Discard the supernatant and resuspend the pellet in 10 ml H2O. Transfer the cells to a 50 ml centrifuge tube.
6. Pellet the cells in a SS34 rotor at 7000 rpm for 5 min.
7. Discard the supernatant and resuspend the pellet in 30 ml sterile 1 X TE/LiAC (made fresh from stocks of 10X TE (0.1 M Tris-HCl, 0.01 M EDTA, pH7.5) and 10X LiAC (1M Lithium acetate adjusted to pH7.5 with dilute acetic acid, Sigma #L6883), and incubate at 30¡C for 15 - 20 min with shaking.
8. Pellet the cells in a SS34 rotor at 7000 rpm for 5 min.
9. Discard the supernatant and resuspend the pellet in 1.5 ml sterile 1X TE/LiAC.
10. Add the plasmid DNAs (5-10 g) and single-stranded carrier DNA (20ug) to a microfuge tube. The carrier DNA is produced by dissolving salmon sperm DNA in TE, sonicating it to reduce its viscosity, extracting it with phenol/chloroform, and precipitating it with ethanol. The DNA is resuspended at a concentration of 5 mg (or 10 mg)/ml in TE, placed in a boiling water bath for 20 min, and immediately cooled on ice. The carrier DNA solution can be stored in aliquots at -20¡C.
11. Add 100ul of the yeast suspension to each microfuge tube.
12. Add 1.2 ml PEG solution (40% PEG, 1X TE, 1X LiAC, made fresh from stocks of 50% PEG 4000, Sigma P3640 3: 350; 10X TE, 10X LiAC) to each tube and incubate at 30¡C for 30 min with sharking.
13. Add dimethyl sulfoxide to 10%. Mix gently.
14. Heat in a 42¡C waterbath for 15 min.
15. Pellet the cells at 7000 rpm for 2 min, aspirate the supernatant, resuspend the cells in 1 ml YEPD, and incubate at 30¡C for 1 hour with shaking.
16. Pellet the cells at 7000 rpm for 5 min and wash once with 1 ml TE.
17. Pellet the cells and resuspend in 0.5 ml TE. Plate 0.1 ml onto each plate (0.5 ml for screening the cDNA library).
18.Incubate the plates at 30¡C until colonies appear.