How do I isolate mitochondria from yeast cells?
Posted February 9, 2024
Answer
The steps for isolating mitochondria from microorganisms are described below.
- Transfer the yeast culture grown overnight into two 15ml tubes in a sterile manner.
- Spin the tubes in a centrifuge at 500g for 10 minutes at 4°C.
- Carefully take out the supernatant without disturbing the pellet.
- Wash the pellet with 1 ml of 0.9% sodium chloride using a micropipette.
- Dispose of the sodium chloride liquid from the tube using a micropipette.
- Mix the pellet with 1 ml of cold lysis buffer using a pipette
- For 10 minutes, let it incubate a shaker at 4°C.
- Centrifuge the mixture at 1000g for 10 minutes at 4°C then remove the supernatant.
- Resuspend the cell pellet in 1.5ml of cold disruption buffer and disrupt the cells using a blunt needle.
- Centrifuge the lysate at 1000g for 10 minutes at 4°C
- Transfer the supernatant to a new tube.
- Centrifuge this supernatant at 6000g for 10 minutes at 4°C and discard the liquid part.
- Wash the remaining pellet with a mitochondria storage buffer.
- Centrifuge the washed pellet at 6000g for 20 minutes at 4°C.
- 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