Yeast Transformation

1. Grow a 25 ml overnight culture to (A₆₀₀ = 1.0)
   • Note: Optimum OD varies for each strain. This is specific for DAY4 a/a .
     
     
2. Centrifuge at 3000 rpm for 2 minutes.
   
   
3. Resuspend the cell pellet in 1 ml of ddH₂O, and recentrifuge for 2 minutes.
   
   
4. Resuspend the cell pellet in 1 ml of 100 mM LiOAc and incubate for 5 min at 30° C.
   
   
5. Place 100m L of cells from previous step into a 1.5 ml tube for each transformation reaction.
   
   
6. Spin the suspension at top speed in a microcentrifuge for 5 sec. Remove the supernatant with a micropipet.
   
   
7. Add the following components into the tube on top of the cell pellet in this order:
   • 240 µl of PEG (50% w/v)
   • 36 µl 1.0 M. LiOAc
   • 5 µl SS-DNA (10.0 mg/ml)
   • 5.0 µl of plasmid DNA (100 ng to 5 µg)
   • 65 µl of sterile ddH₂O.
   • Note: The order is important. The PEG shields the cells from the high concentration of LiOAc.
     
     
8. Vortex the cell pellet for at least 1 min to resuspend the cell pellet in the transformation mix and incubate at 42° C for 15 minutes.
   • Note: The optimum heat shock time varies with yeast strain. (15 minutes is for DAY4 a/a )
     
     
9. Pellet the cells at top speed in a microcentrifuge for 10 sec. Remove the supernatant using a micropipet.
   
   
10. Gently resuspend the pellet in 200 - 400 µl of sterile ddH₂O by slowly pipetting up and down.
    • Note: Be as gentle as possible at this step if high efficiency is important.
      (if you are getting too many colonies, add 1 ml of sterile ddH₂O instead and plate 500µl on 2 plates)
      
      
11. Plate the cell suspension onto 1 or 2 plates of SC omission medium that selects for the presence of the plasmid. Let the plate sit for a little so that it would absorb the mixture. Colonies should be visible in 2 - 4 days at 30^o C.