Transformation of S.

cerevisiae

S. cerevisiae is grown at 30 degrees. The phase of growth of your cells is very important. Cultures that have reached stationary phase transform very poorly. Pipet slowly and carefully, these reagents are very viscous!

Cell growth
1. The day before you intend to do the transformation, inoculate a 5 mL culture of cells in liquid YPD media and place on the rotating wheel at 30 degrees. 2. Approximately 4 hours before you intend to do the transformation, dilute the overnight culture into 50 mL of YPD at a density of 5 x 106 cells/mL. 3. Grow the culture at 30 degrees for 4 hours, then proceed as below.

Washing cells
4. Pellet cells by spinning 5 minutes at 2500 rpm in the tabletop centrifuge. 5. Discard supernatant. 6. Add 25 mL sterile water to tube with cells. 7. Resuspend cells by vortexing. 8. Pellet cells by spinning 5 minutes at 2500 rpm in the tabletop centrifugte. 9. Discard supernatant, saving cell pellet. 10. Resuspend pellet in 1 mL sterile water by vortexing. 11. Transfer cells to a microfuge tube 12. Pellet cells 2 minutes in personal microfuge 13. Resuspend cell pellet in 1 mL sterile water. 14. For each transformation, pipette 100 uL of cell suspension into microfuge tube. 15. Spin down cells for 2 minutes in your personal minicentrifuge. 16. Remove supernatant liquid, leaving behind cells. 17. Add transformation reaction mixes in the following order: 275 ul transformation mix (contains 240 ul 50% polyethylene glycol, 35 ul 1.0 M lithium acetate) 50 ul salmon sperm carrier DNA (2 mg/mL, keep on ice, boiled before use: see note) 33 ul DNA plus water

18. Vortex to thoroughly mix cells and transformation reagent. 19. Heat shock cells by placing in 42 degree water bath for 45 minutes. At the end of the heat shock, spin transformation reactions for 1 minute to pellet cells. 20. Remove transformation mix with a pipet, leaving behind cell pellet.

21. Add 1000 ul (1 mL) YPD medium to each transformation. Resuspend cells by gently pipetting up and down and stirring gently with your pipet tip. 22. Place tubes containing transformations in empty test tubes on the rotating wheel at 30 degrees for 1 -3 hours. During this time the cells begin to express the drug resistance gene, so that they will be able to survive the drug. (If youre selecting for an auxotrophic marker, the resuspended cells can be plated directly without recovery.) 23. Pipet 100-200 ul of transformed cells onto plate and spread out the transformation. 24. Incubate the plate at 30 degrees in an inverted position for 2-4 days until colonies appear.

Note on salmon sperm DNA:

It is not necessary or desirable to boil the salmon sperm carrier DNA each time. Keep an aliquot in your own freezer box. Every three to four freeze/thaws, place DNA at 95 degrees for 5 minutes, then place on ice. The carrier DNA should be kept on ice at all times.

Adapted from Gietz, R.D. and R.A. Woods. (2002) TRANSFORMATION OF YEAST BY THE Liac/SS CARRIER DNA/PEG METHOD. Methods in Enzymology 350: 87-96.