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AAT Bioquest

How do I isolate mitochondria from yeast cells?

Posted February 9, 2024


Answer

The steps for isolating mitochondria from microorganisms are described below. 

  1. Transfer the yeast culture grown overnight into two 15ml tubes in a sterile manner. 
  2. Spin the tubes in a centrifuge at 500g for 10 minutes at 4°C. 
  3. Carefully take out the supernatant without disturbing the pellet. 
  4. Wash the pellet with 1 ml of 0.9% sodium chloride using a micropipette. 
  5. Dispose of the sodium chloride liquid from the tube using a micropipette. 
  6. Mix the pellet with 1 ml of cold lysis buffer using a pipette
  7. For 10 minutes, let it incubate a shaker at 4°C. 
  8. Centrifuge the mixture at 1000g for 10 minutes at 4°C then remove the supernatant. 
  9. Resuspend the cell pellet in 1.5ml of cold disruption buffer and disrupt the cells using a blunt needle.
  10. Centrifuge the lysate at 1000g for 10 minutes at 4°C
  11. Transfer the supernatant to a new tube. 
  12. Centrifuge this supernatant at 6000g for 10 minutes at 4°C and discard the liquid part.
  13. Wash the remaining pellet with a mitochondria storage buffer. 
  14. Centrifuge the washed pellet at 6000g for 20 minutes at 4°C. 
  15. Resuspend the final pellet in the mitochondria storage buffer and store it at -20°C.
Additional resources

Isolation of Mitochondria from Plants, Yeast Cells, Mice, Cell Culture

Mitochondria

MitoLite™ Blue FX490