Transformation of Pichia pastoris
Materials
- 150 µL competent Pichia Pastoris (- 80 °C)
- Plasmid of interest
- EcoRV restriction enzyme
- Sodium Acetate (3 M)
- EtOH abs.(ice cold, -20 °C)
- 70% EtOH (ice cold, -20 °C)
- Ice bath
- Electroporation cuvette
- Electroporation device
- Sorbitol (1 M), ice cold, -20 °C
- YPD medium (yeast extract (10 g/L), peptone (20 g/L), glucose (20 g/L))
Procedure
- Linearize 20 µg of plasmid of interest with EcoRV
- Purify via EtOH precipitation:
- Adjust sample volume to 50 µL (MilliQ)
- Add 5 µL sodium acetate, vortex
- Add 125 µL ice cold EtOH abs.; Incubate on ice for 15 min
- Centrifuge 30 min, 14’000 rpm, 4 °C
- Discard the supernatant
- Wash the pellet with 500 µL colt EtOH 70 %
- Centrifuge 15 min, 14’000 rpm, 4 °C
- Remove the supernatant, dry pellet at RT
- Dissolve in 15-25 µL MilliQ
- Thaw competent Pichia on ice
- Add 10 µg linearized plasmid
- Incubate on ice for 5 min
- Pipet into electroporation cuvette on ice
- Perform electroporation
- 1500 V, 125 µF, 200 Ω
- Adjust all settings
- Insert cuvette into apparate
- Press both red buttons until tone sounds
- Immediately add 500 µL ice cold sorbitol
- Add 500 µL YPD medium
- Grow 1 h at 30 °C, 200 rpm
- Plate 100 µL undiluted suspension and 1:10 dilution of YPD agar plates with G418; grow at 30°C
- Keep only the big colonies for testexpression