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))

- YPD agar plate with G418 (500 mg/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