Oct 20, 2019
Pichia pastoris transformation through electroporation
- 1Heinrich-Heine Universität Düsseldorf
Protocol Citation: Igem Dusseldorf 2019. Pichia pastoris transformation through electroporation. protocols.io https://dx.doi.org/10.17504/protocols.io.8heht3e
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 20, 2019
Last Modified: October 20, 2019
Protocol Integer ID: 28934
Materials
linearized vector DNA with electrocompetent Pichia cells
1 mL of 1:1 1 M sorbitol
YPD Liquid Medium or Plates (500 ml):
- 5 g yeast extract
- 10 g peptone
- 10 agar (if preparing plates)
- Fill to 450 mL with water
-> autoclave
YPDS + antibiotic Plates (1 liter):
- 10 g yeast extract
- 20 g peptone
- 182.2 g sorbitol
- Fill to 800 mL with water
- Mix until dissolved
- Transfer 400 mL two 1 L bottles
- pre-filled with 10 g BactoAgar
- Fill each bottle to 450 mL with water
--> autoclave
- Cool bottles to ~60°C, add 50 mL of 10x D
- Mix well, then aliquot in 4 bottles of 250 mL
Prior
Prior
Prior to transformation plasmid DNA containing the gene(s) of interest was linearized in
restriction digests using an enzyme that only cuts once following the manufacturers manual !
Transformation
Transformation
Mix approx. 150 ng of linearized vector DNA with electrocompetent Pichia cells
Tranfer mixture to ice-cold 0.2 cm electroporation cuvette
Incubate cuvette for 2 min
Electroporate using a BioRad MicroPulser with a charging voltage of 1.5 kV
Immediately add 1 mL of 1:1 1 M sorbitol and YPD (v/v) to recover the cells
Transfer mixture to a plastic culture tube and incubated at 30°C for 1 hour at 225 rpm.
Centrifuge culture tube at 3.000 x g for 5 min to pellet the cells
Resuspend pellet in 200 μL YPD media
Plate out cells on YPDS plates containing desired antibiotic and incubated at 30°C for 3 days