Grow cells over night in 50 ml of appropriate medium to an OD600 of 0.3 to 1.5.

↓ 3200 rpm, 5 min

Remove supernatant. Wash with 50 ml d.w.

↓ 3200 rpm, 5 min

Remove supernatant. Wash with 10 ml LiSorb.

↓ 3200 rpm, 5 min

Remove supernatant and resuspend cells in 300 ul of LiSorb.

↓

Aliquot cells in 50 ul portions. Add 5 ul of denaturated 10 mg/ml carrier DNA (heat 5 min at 95ﾟC to denature DNA, place on ice for a couple of seconds).

↓

Mix cells and incubate for 20 min at RT.

↓

Add 35 ul DMSO. Vortex. Heat shock cells for 5-15 min at 42ﾟC. (Wild type: 15 min, ts-mutants: 5 min.)

↓ 1500 rpm, 3 min, 4ﾟC

Remove supernatant. Add 300 ul appropriate medium or PBS (steril).

↓

Resuspend and plate out on selective plate.

(For G418-selection: the cells were resuspended in 3 ml of liquid YPDA, shaked for 3 hrs at 30ﾟC, before spreading them on slective G418 plates. Hygromycin for 8 hrs.)