Generation of competent Pichia pastoris: Difference between revisions

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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..."
 
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===== 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

Revision as of 14:05, 5 June 2025

Materials
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 108 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
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