Day before

1. Pick colony from plate into 5mL growth media for overnight growth in 30°C shaker

Lithium Acetate Prep

Cell prep

2. Back dilute over night culture to OD 0.175 in 15mL growth media.

3. Grow up yeast to OD600 ~0.75 at 30°C. Takes 4-5 hours for WT in YPD.

4. Pellet at 2000 rpm for 10 min in large centrifuge.

5. Prepare lithium acetate transformation while harvesting cells (see table).

6. Wash with 0.5x volume H₂O and repeat step 4.

7. Wash with 0.5x volume 100mM LiOAc and repeat step 4.

8. Final suspension in 0.01x volume 100mM LiOAc (100 μL per transformation - approx 300µL from 15mL culture).

9. Pellet at 8000 rpm for 1 min at 20°C on small tabletop centrifuge.

Heat Shock Procedure

10. Resuspend in the lithium acetate transformation mix. I suggest resuspending with the plasmid DNA and ddH₂O (54 μL) first, then mixing with the LiOAc+PEG+ssDNA mixture (306 μL) afterwards, as it is too viscous to easily disrupt the pellet.

11. Incubate 30°C for 30 min.

12. Incubate for a further 17 min at 42°C in water bath/heat block.

13. Centrifuge 8000 rpm for 2 min and remove supernatant.

14. Depending on the selection marker:

a. Prototrophic/auxotrophy gene: Resuspend in 200 μL of H₂O.

b. Eukaryotic antibiotic: Recover in 1.0mL YPD for 2-3 hrs at 30°C.

15. Plate desired volume on plates. Typically do half.

Transformation of Integration Cassettes

16. NotI digest

- approx. 1 μg of cassette

- 2µL NEBuffer 3.1 (NotI) or CutSmart (NotI-HF)

- 0.2µL NotI (NEB)

- up to 20 μL H₂O

17. 1h incubation at 37°C

18. Optional PCR clean up for better results

19. Transform the entire mixture