Transformation of Pichia pastoris - Revision history

ESO wikiadmin at 13:10, 5 June 2025

2025-06-05T13:10:11Z

← Older revision		Revision as of 15:10, 5 June 2025
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	- Sorbitol (1 M), ice cold, -20 °C<br>		- Sorbitol (1 M), ice cold, -20 °C<br>
	- YPD medium (yeast extract (10 g/L), peptone (20 g/L), glucose (20 g/L))<br>		- YPD medium (yeast extract (10 g/L), peptone (20 g/L), glucose (20 g/L))<br>
	- YPD agar plate with G418 (500 mg/L)}}		- YPD agar plate with G418 (500 mg/L)\|titel=Materials}}

	== Procedure ==		== Procedure ==

ESO wikiadmin at 13:09, 5 June 2025

2025-06-05T13:09:50Z

← Older revision		Revision as of 15:09, 5 June 2025
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	- Plasmid of interest<br>		- Plasmid of interest<br>
	- EcoRV restriction enzyme<br>		- EcoRV restriction enzyme<br>
	- Sodium Acetate (3 M)		- Sodium Acetate (3 M)<br>
	- EtOH abs.(ice cold, -20 °C)		- EtOH abs.(ice cold, -20 °C)<br>
	- 70% EtOH (ice cold, -20 °C)		- 70% EtOH (ice cold, -20 °C)<br>
	- Ice bath		- Ice bath<br>
	- Electroporation cuvette		- Electroporation cuvette<br>
	- Electroporation device		- Electroporation device<br>
	- Sorbitol (1 M), ice cold, -20 °C		- Sorbitol (1 M), ice cold, -20 °C<br>
	- YPD medium (yeast extract (10 g/L), peptone (20 g/L), glucose (20 g/L))		- YPD medium (yeast extract (10 g/L), peptone (20 g/L), glucose (20 g/L))<br>
	- YPD agar plate with G418 (500 mg/L)}}		- YPD agar plate with G418 (500 mg/L)}}

ESO wikiadmin at 13:09, 5 June 2025

2025-06-05T13:09:09Z

← Older revision		Revision as of 15:09, 5 June 2025
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			{{Protokoll-Mix\|Inhalt=- 150 µL competent Pichia Pastoris (- 80 °C)<br>
			- Plasmid of interest<br>
			- EcoRV restriction enzyme<br>
			- 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 ==		== Procedure ==

ESO wikiadmin: Created page with " == 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 Mil..."

2025-06-05T13:07:54Z

Created page with " == 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 Mil..."

New page

== 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