Yeast transformation

Back to index

Notes

-If you are doing fewer than 5 transformations, you can cut the volumes in steps 2-4 and 7 in half.

Protocol

1. The day before the transformation, inoculate 3 mL YPD or SC medium with a single colony. Grow overnight at 30C.

2. In the morning, dilute the overnight culture to OD600 = 0.3-0.4 in 50 mL YPD or SC medium. Grow 50 mL culture until OD600 = 1.0-1.2 (this should take 4-5 hr).

3. Pellet cells in a 50 mL conical tube at 3,000 x g for 3 min. While cells are spinning, heat an aliquot of 2 mg/mL salmon sperm DNA at 95C.

4. Wash cells with 50 mL H₂O and pellet as above.

5. Resuspend cells in 1 mL H₂O and transfer to a microfuge tube. Flash spin to pellet cells.

6. Place denatured ssDNA in an ice/water bath.

7. Resuspend cells in 1 mL H₂O, transfer 100 μL aliquots to new microfuge tubes, and pellet as above.

8. Prepare the following transformation mix for each sample, plus one extra:

   	1 transformation	5 (+1) transformations
   50% w/v PEG-3350	240 μL	1440 μL
   1 M LiAc	36 μL	216 μL
   Boiled 2 mg/mL ssDNA	50 μL	300 μL
   DNA + H₂O	34 μL	204 μL
   Total	360 μL	2160 μL

9. Add 360 μL transformation mix to each cell pellet and resuspend by vortexing.

10. Incubate the samples at 42C for 40 min.

11. Pellet cells at 6,000 x g for 30 sec.

12. Resuspend cells in 200 μL H₂O and plate.