ESO wikiadmin at 13:20, 5 June 2025

2025-06-05T13:20:20Z

← Older revision		Revision as of 15:20, 5 June 2025
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			{{Protokoll-Mix\|titel=Materials\|Inhalt=These reagents are important:
			*YPD – medium (yeast extract (10 g/L), peptone (20 g/L), glucose (20 g/L))
			*BMMY medium (yeast extract (10 g/L), peptone (20 g/L), yeast nitrogen base
			(13,4 g/L), potassium phosphate pH 6.0 (0,1 M), biotin (0.4 mg/L), methanol (0,5 %))
			*MeOH abs.
			*Transformed Pichia clones
			*50 mL falcons
			*Spectrometer for OD measurments
			*Cuvettes
			*1,5 mL Eppis‘
			*PBS
			*4x SDS loading buffer}}

	== Procedure ==		== Procedure ==

ESO wikiadmin: Created page with " == Procedure == * Inoculate 5 mL YPD medium with one '''big''' colony ** in 50 ml falcon tube (secure lid with tape, don’t tighten lid) * Grow o. n. 30 °C, 200 rpm * Centrifuge at 2000 g, 5 min * Resuspend the complete pellet in 5 mL BMMY medium * Measure the OD ** Use a 1:100 dilution for measurement * Start growth For starters you don’t need to dilute your suspension For optimization purposes you may want to adjust your starting OD *** Recommended is a sta..."

2025-06-05T13:17:50Z

Created page with " == Procedure == * Inoculate 5 mL YPD medium with one '''big''' colony ** in 50 ml falcon tube (secure lid with tape, don’t tighten lid) * Grow o. n. 30 °C, 200 rpm * Centrifuge at 2000 g, 5 min * Resuspend the complete pellet in 5 mL BMMY medium * Measure the OD ** Use a 1:100 dilution for measurement * Start growth ** For starters you don’t need to dilute your suspension ** For optimization purposes you may want to adjust your starting OD *** Recommended is a sta..."

New page

== Procedure ==

* Inoculate 5 mL YPD medium with one '''big''' colony
** in 50 ml falcon tube (secure lid with tape, don’t tighten lid)
* Grow o. n. 30 °C, 200 rpm
* Centrifuge at 2000 g, 5 min
* Resuspend the complete pellet in 5 mL BMMY medium
* Measure the OD
** Use a 1:100 dilution for measurement
* Start growth
** For starters you don’t need to dilute your suspension
** For optimization purposes you may want to adjust your starting OD
*** Recommended is a starting OD between OD 3 and OD 40
* Grow at 30 °C, 200 rpm
* Take samples of 200 µL and add 25 µL MeOH after 24 h, and 48 h
** Sample handling
*** Measure the OD (1:100 dilution recommended)
*** Centrifuge 5 min, 2000 rpm
*** Split into pellet and supernatant
*** Resuspend the supernatant in 200 µL PBS
*** Mix supernatant with 4x SDS loading buffer
*** Mix equivalent of OD 1 from pellets with 80 µL of 4x SDS loading buffer
* End the testexpression after 72 h, take samples

For optimization, you may want to try different methanol concentrations between 0,5 % and 1.5 % MeOH

Testexpression in Pichia pastoris - Revision history

ESO wikiadmin at 13:20, 5 June 2025