Transformation by the lithium acetate (LiOAc) method (Ito et al., 1983; Gietz and Shiestl, 1991).

Day 1 (0,5 hours in the morning (step 1); 0,5 hours in the evening (steps 2-3):

1. Inoculate 4 ml YPD medium with the appropriate culture
   Incubate at 30°C
2. Determine cell density of the preculture by measuring OD₆₀₀ and counting the cells
3. Resuspend cells from the preculture in 50 ml YPD at a final concentration of 10⁴ cells/ml. (cfr. Table in section 0)
   Incubate overnight (± 16 hours) at 30°C to a density of OD₆₀₀ 0,7 to 1,0 (approximately 10⁷ cells/ml)

Day 2 (3 hours in the morning):

1. Determine cell density of the preculture by measuring OD₆₀₀ and counting the cells
   Concentration of about 10⁷ cells/ml is required
2. Harvest cells by centrifugation in Sigma 3K30 at 4.000 rpm for 5 min
3. Resuspend pellet in 20 ml of sterile milliQ water
   Centrifuge in Sigma 3K30 at 4.000 rpm for 5 min
4. Resuspend cell pellet in 1,5 ml of freshly prepared sterile LATE
   Incubate for 1 hour at 30°C with constant agitation
   Transfer cell suspension into a 1,5 ml Eppendorf tube
   Harvest cells by centrifugation (microcentrifuge, 5.000 rpm for 1 min)
5. Resuspend cell pellet in 200 µl of freshly prepared sterile LATE
   Cell concentration about 2.10⁹ cells/ml
   
   This suspension is stored on ice until being transformed.

1. Boil a stock sollution of fish sperm DNA for 10 min in a water bath
   Chill in icewater
2. Mix 25 µl of freshly denatured fish sperm DNA with 0,1 µg of plasmid DNA and 50 µl of yeast cells in LATE
   Mix gently (no vortexing !)
   Incubate for 0,5 hours at 30°C with constant agitation
3. Add 300 µl of freshly prepared sterile PLATE
   Mix carefully (no vortexing !)
4. Incubate cells for 30 min at 30°C with constant agitation
5. Incubate cells for 15 min at 42°C
6. Add 800 µl of sterile water
   Mix gently
   Collect cells by centrifugation (microcentrifuge, 5.000 rpm for 1 min)
7. Resuspend cells in 500 µl TE
   Plate 50 µl and 450 µl onto selective YPD plates or onto complete minimal drop-out medium
8. Incubate plates at 30°C for 2 - 3 days, until colonies appear

• 10x TE (100 mM Tris.HCl; 10 mM EDTA; pH 7,5)
• 10x LiOAc (1M LiOAc adjusted to pH 7,5 with dilute acetic acid)

• 8 vol of 50% PEG 4000 - PEG 3350
• 1 vol 10x TE
• 1 vol 10x LiOAc

Fish sperm DNA (Boehringer; 10 mg/ml): stored at -20°C

50 ml YPD in 250 ml erlenmeyer

1 empty sterile 4 ml glass tube

Sigma 3K30 centrifuge

Microcentrifuge

Heat block / water bath at 95°C

Heat block / water bath at 42°C

Selective YPD plates or complete minimal drop-out plates (2/transformation)