ELECTROPORATION OF Pichia pastoris

Competent cells

1.  Inoculate a 5 ml o/n culture in YPD with a single colony (or from glycerol stock). Grow at 30C at 300rpm.

2.  Next day, inoculate a 500 ml YPD culture with 50-100 μl of the overnight around 5 pm. Grow o/n at 30C at 300rpm.

3.  Next day, harvest cells when they reach an OD600 of 1.3-1.5. Spin down cells in 2 x 250 ml canisters using the swinging bucket rotor (JS 5.3) at 2000xg for 5 minutes. All rotors are at 4C.

4.  Pour off media and resuspend each pellet in 50 ml of YPD and add 10 ml 1 M HEPES pH 7.8, and 1.25 ml 1 M DTT to each pellet. Let stand 15 minutes at 30C. Add ice cold H2O to 250 ml (each canister).

5. From this point on, everything should be at 4C or on ice. 

6. Spin cells at 2000xg for 5 minutes.

7. Resuspend cell pellets in a total volume of 250 ml ice cold H2O. Combine pellets into one canister.

8. Spin cells at 2000xg for 5 minutes.

9. Resuspend pellet in 20 ml of ice cold 1 M sorbitol and transfer cells to Oakridge tube.

10. Spin cells at 2000xg for 5 minutes using the JA-17 rotor.

11. Resuspend cell pellet in 0.5-0.75 ml of ice cold 1 M sorbitol. Final volume will be 0.8-1.5 ml.

Electroporation

1.  Use 40-80 μl of cells and add to the eppendorf tube containing 5-10 μl PmeI-linearized plasmid in H2O. Let sit on ice for 3 minutes.

2. Transfer to an ice cold Bio-Rad electroporation cuvette (0.2 cm) and let sit on ice for 10-15 minutes.

3. Electroporate with Bio-Rad gene pulser (exponential setting). Settings: 1500 V, 200 Ώ, 25 μF. The rate constants should be 4.4-4.8 or so.

4. Immediately add 1 ml of ice cold 1 M sorbitol to the cuvette and transfer cells to a 15 ml tube.

5. Let stand at 30C for 60 minutes without shaking.

6. Add 1 ml of prewarmed YPD and shake at 250 rpm at 30C.

7. Plate various amounts of cells on YPDS plates containing zeocin. There is not much point in plating very large volumes of cells (i.e. don’t plate more than 200 μl). They will make a lawn and there will be few discrete colonies.

8. Incubate plates at 30C for 2-6 days. Put a beaker of water in the incubator to keep plates from drying out.