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Isolation of Mitochondria
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Materials Required

 

  1. Yeast culture.
  2. Refrigerated Centrifuge.
  3. 15ml centrifuge tubes.
  4. Sodium Chloride (0.9%).
  5. Micropipette.
  6. Ice cold lysis buffer.
  7. Shaker.
  8. Mitochondria storage buffer.
  9. Refrigerator.
  10. 15ml micro centrifuge tube.

 

Procedure

 

  1. Aseptically transfer the overnight yeast culture into two 15ml centrifugation tubes.
  2. Centrifuge it at 500g for 10 minutes at 4oC.
  3. Carefully remove the supernatant without disturbing the pellet.
  4. Carefully rinse the pellet in 1ml sodium chloride (0.9%) using a micropipette.
  5. The sodium chloride from the centrifugation tube is discarded using a micropipette.
  6. Resuspend the pellet in 1ml of ice cold lysis buffer and mix well using a micropipette.
  7. Incubate it at 4oC on a shaker for 10 minutes.
  8. Centrifuge it at 1000g for 10 minutes at 4oC and carefully remove the supernatant.
  9. Resuspend the cell pellet in 1.5 ml ice cold disruption buffer and complete cell disruption by using the blunt end of a needle.
  10. Centrifuge the lysate at 1000g for 10 minutes at 4oC.
  11. Transfer the supernatant to a fresh 15mL tube and also mix the supernatant obtained from the step 7.
  12. Centrifuge it at 6000g for 10 minutes at 4oC and discard the supernatant.
  13. Wash the pellet with mitochondria storage buffer.
  14. Centrifuge it at 6000g for 20 minutes at 4oC.
  15. Resuspend the pellet in mitochondria storage buffer and store at -20oC.

 

Difference Encountered in a Real Laboratory


In an actual laboratory setting, there are certain important steps that are not necessarily applicable in a virtual lab:


1.    Before starting the experiment sterile the laminar air flow chamber using spirit.
2.    All the tubes and bottles used in the experiment should labeled properly.
3.    Always disinfect your work area when you are finished.
 

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