Isolation of Mitochondria

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In this lab, we used differential centrifugation to isolate mitochondria and nuclei from cauliflower. We also measure the rate at which a certain molecule is converted to another molecule and how much a given enzyme can convert at a given time. Then we will get three fractions and find out which fraction has the most mitochondria and which fraction has the purest mitochondria. Nuclei and mitochondria are both organelles that are found within most eukaryotic cells. The nucleus is enclosed by a double membrane nuclear envelope. Inside the nucleus DNA is organized into material called chromatin. Nuclei are the central part of the cell, the densest organelle, and considered to be the brain of the cell. Mitochondria are membrane-bound organelles, and like the nucleus have a double membrane. The outer membrane is fairly smooth. But the inner membrane is highly intricate, forming folds called cristae. The cristae greatly increase the inner membrane's surface area. Mitochondria are cellular power plants because they generate most of the cell's supply of adenosine triphosphate (ATP), used as a source of chemical energy. During the lab, we will not be able to see the mitochondria, so we completed an enzyme assay for succinate dehydrogenase to report the mitochondria. In preparing three cell fractions, we needed to weigh out 30 g. of chilled cauliflower and cut it into little pieces. Then grind it up in a chilled mortar with 30 ml of cold grinding solution. While grinding the cauliflower, we sprinkle some cold sand into the mortar then poured the solution through 4 layers cheese cloth into a clean cold beaker. Then pour the filtrate into a chilled centrifuge and centrifuge at 1000 x g for 10 minutes at 4°C. During the centrifuge, we prepared 7 cuvettes for enzyme assay. Each cuvette has 0.5 ml of 0.3 M phosphate buffer, 0.5 ml of 0.05 M MgSO4, 0.5 ml of 0.06 M HCl and 4 drops

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