Yeast transformation from a liquid culture

1. Grow yeast in 250 ml YEPD until A600 is 0.2-0.8. There are two ways to do this:
   • Inoculate the YEPD with a patch of yeast in the afternoon and grow O/N at room temperature.
   • Inoculate the YEPD with an overnight culture to A600 of ~0.1 and grow at 30°C for ~4hrs.
2. Before starting:
   • boil salmon sperm DNA (10µg/µl) for 5 min an put on ice.
   • prepare TE/LiAc
   • prepare TE/LiAc/PEG
3. Spin at 2000 rpm, 20°C for 5 min.
4. Resuspend in 50ml sterile H2O at RT.
5. Spin at 2000 rpm, 20°C for 5 min.
6. Resuspend in 10ml TE/LiAc at RT.
7. Spin again.
8. Resuspend in TE/LiAc in a total volume of 1 ml per 0.1OD.
9. To 20µl of cells, add 2µl Salmon sperm DNA.
10. Transfer 20µl to each well of a U-bottom 96-well plate.
11. Add 8µl of plasmid DNA (~0.5µg).
12. Add 100 µl TE/LiAc/PEG solution and resuspend carefully.
13. Incubate at 30°C for 30 min.
14. Incubate at 42°C for 15 min.
15. Centrifuge 5 min at 1800 rpm at RT.
16. Remove the supernatant with a multichannel pipette.
17. Add 110µl of sterile water, and directly remove 100µl.
18. Resuspend cells in the remaining water and spot the cells on a plate.
19. Replica-clean the next day if necessary.