YEAST TRANSFORMATION PROTOCOL
for Library Screening

Erickson lab, 2011

Starter yeast culture: Inoculate 30-50 ml of liquid medium with 5-8 colonies of the yeast strain and incubate 1-2 days at 225 rpm and 30°C. Use SCD –trp +leu media for yeast strain AH109  containing a bait plasmid containing TRP1 gene marker.

1. In a sterile 1 or 2 liter flask, add 15-20 ml of saturated overnight culture to 150 ml of 2x YPAD media to produce an OD₆₀₀ between 0.15 – 0.30

2. Incubate culture at 30°C and 225 rpm for 4-6 hours to obtain an OD₆₀₀ between 0.40 and 0.60. For high transformation efficiency cells need to be in mid-log growth phase (OD < 0.70).

3. Transfer culture to sterile 50 ml tubes and pellet cells using centrifugation at 2500 rpm for 5 min; gently resuspend each tube of cells in ~25 ml of sterile water or TE; pool cells, re-spin, and gently resuspend cells using a total of 0.75 ml of 0.1M LiOAc/TE. (0.25 ml per 50 ml of original culture)

4. Assemble transformation reaction in 50 ml tube (in this order):

· 50-100 µg library plasmid

· 2 mg boiled carrier-DNA (200 ul of 10 mg/ml boiled herring or salmon sperm DNA)

· 1 ml yeast cells

· Short vortex to mix

· 6 ml LiOAC/TE/PEG (make fresh each time. 6 ml stock = 0.6 ml 1M LiOAC + 0.6 ml 10X TE + 4.8 ml 50% PEG₃₅₀₀)

· Short vortex to mix

5. Incubate reactions at 30°C and 200 rpm for 30 minutes.

6. Add 700 µl of DMSO to transformation mixture. Mix gently by inverting tube, don’t vortex.

7. Heat shock: Incubate tubes in a 42°C water bath for 15 minutes with occasional swirling.

8. Pellet cells: Spin at 2.5K rpm in a for 5 minutes; pour of transformation mix and remove remaining liquid with a micropipettor.

9. Gently resuspend cell pellet using 3-5 ml of YPAD media; stir the pellet by with a blue micropipette tip to gently resuspend-do not vortex.

10. Plate 150 ul of cell suspension onto SCD (-trp - leu –ade -his) selection plates. Spread cells gently using minimal number of passes with cell spreader. Let plates dry for a few minutes with lids slightly ajar. Pipet 2 µl of cells into a 100 ul puddle of sterile water on a SCD-t-l plate to obtain transformation efficiency.

11. Incubate dried plates upside down at 30°C. Tiny colonies should be seen in 2 days on SCD-t-l plates. Colonies will begin to appear after 4 days on SCD-t-l-a-h plates. If too many colonies appear in 4-8 days, one should have added 3-AT to plates.