Generation of competent Pichia pastoris

Materials

Wildtype Pichia Pastoris yJC100, grown on YPD agar plate

• YPD medium (yeast extract (10 g/L), peptone (20 g/L), glucose (20 g/L))
• Transformation buffer (Tris (10 mM) pH 7,5, Sorbitol (0,6 M), LiAc (100mM), DTT (10 mM))
• Sorbitol (1M)
• Inoculation loop
• Erlenmeyer flask 500 mL
• Spectrometer for OD measurement
• 50 mL falcons

Procedure

• Inoculate 5 mL of YPD medium with one big colony
• Grow 8 h at 30 °C, 200 rpm
• Inoculate 100 mL YPD medium with 1 mL preculture
• Grow o.n. at 30 °C, 200 rpm
• Measure the OD
• Aliquot 15 x 10⁸ cells, centrifuge 5 min, 1500 g
• Resuspend in 30 mL transformation buffer each
• Incubate at RT for 30 min
• Centrifuge 5 min, 4°C, 1500 g
• Resuspend in 5 mL Sorbitol each
• Repeat 3x
• Resuspend in 320 µL Sorbitol
• Freeze down in 150 µL aliquots
• Store at -80 °C