Generation of competent Pichia pastoris - Revision history

ESO wikiadmin at 13:05, 5 June 2025

2025-06-05T13:05:42Z

← Older revision		Revision as of 15:05, 5 June 2025
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	*50 mL falcons}}		*50 mL falcons}}

	~~===~~== Procedure ==~~===~~		== Procedure ==

	* Inoculate 5 mL of YPD medium with one big colony		* Inoculate 5 mL of YPD medium with one big colony
	* Grow 8 h at 30 °C, 200 rpm		* Grow 8 h at 30 °C, 200 rpm

ESO wikiadmin at 13:05, 5 June 2025

2025-06-05T13:05:25Z

← Older revision		Revision as of 15:05, 5 June 2025
Line 9:		Line 9:

	===== Procedure =====		===== Procedure =====
	~~- Inoculate 5 mL of YPD medium with one big colony~~

	- Grow 8 h at 30 °C, 200 rpm		* 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		* Inoculate 100 mL YPD medium with 1 mL preculture
			* Grow o.n. at 30 °C, 200 rpm
	- Grow o.n. at 30 °C, 200 rpm		* Measure the OD
			* Aliquot 15 x 10<sup>8</sup> cells, centrifuge 5 min, 1500 g
	- Measure the OD		* Resuspend in 30 mL transformation buffer each
			* Incubate at RT for 30 min
	- Aliquot 15 x 10<sup>8</sup> cells, centrifuge 5 min, 1500 g		* Centrifuge 5 min, 4°C, 1500 g
			* Resuspend in 5 mL Sorbitol each
	~~- Resuspend~~ in 30 mL transformation buffer each		* Repeat 3x
			* Resuspend in 320 µL Sorbitol
	~~- Incubate~~ at RT for 30 min		* Freeze down in 150 µL aliquots
			* Store at -80 °C
	~~- 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

ESO wikiadmin: Created page with "{{Protokoll-Mix|Inhalt=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..."

2025-06-05T13:04:45Z

Created page with "{{Protokoll-Mix|Inhalt=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..."

New page

{{Protokoll-Mix|Inhalt=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 10<sup>8</sup> 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