Editing Pichia EXpression (section)

=== Competent Cells  ===

1: Grow a 5 ml overnight culture of P. or K. pastoris cells in YPD in a 30°C shaking incubator.<br>2: The next day, dilute the overnight culture to an A600 of 0.15 to 0.20 in a volume of 50 mL of YPD in a flask large enough to provide good aeration. (Starting volumes may be scaled up or down)<br>3: Grow yeast to an A600 of 0.8-1.0 in a 30°C shaking incubator, approximately 4-5 hours.<br>4: Centrifuge the culture at 500 x g for 5 minutes at room temperature and pour off the supernatant.<br>5: Resuspend the pellet in 9 ml of ice-cold BEDS solution, supplemented with 1 ml of 1M Dithiothreitol (DTT).<br>6: Incubate the cell suspension for 5 minutes at 100 rpm in the 30°C shaking incubator.<br>7: Centrifuge the culture again at 500 x g for 5 minutes at room temperature and resuspend the pellet in 1 ml of BEDS solution without DTT.<br>8: The cells are now ready for transformation. Alternatively, they may also be stored at -80°C&nbsp; after slowly freezing them by placing the aliquots of 200 μL volume in a Styrofoam box placed in the -80°C freezer overnight. '''Competent cells can be stored at least 6 months at -80 oC'''.<br><br>