Mitochondria lab activity
Answers
Explanation:
Mitochondria: the energy converters
Mitochondria, using oxygen available within the cell convert chemical energy from food in the cell to energy in a form usable to the host cell. The process is called oxidative phosphorylation and it happens inside mitochondria.
The mitochondrion is often referred to as the powerhouse of the cell. It contains all of the machinery needed to provide the cell and its components with energy to carry out cellular processes.
Isolate mitochondria – grind 20g cauliflower in chilled mortar with 40 mL cold mannitol grinding medium and 5 g cold purified sand for 4 minutes.
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Filter suspension with cheesecloth, centrifuge at 600 g at 4° C for 10 minutes.
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Centrifuge postnuclear supernatant fluid at 10,000 g at 4 C for 30 minutes.
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Discard postmitochondrial supernatant fluid, add 7.0 mL cold mannitol assay medium to mitochondrial pellet.
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Scrape pellet from tube, resuspend sediment in assay medium with pipet (make sure clumps are completely dispersed). Keep in ice bath.
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Label 10 cuvettes based on the following table from page 135 of lab manual. Heat 0.6 mL of suspension for tube 7 in boiling water for 5 min, then cool in ice bath. Set spectrophotometer at 600 nm.
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Add correct volume of assay medium and various solutions indicated from the table, but don’t add mitochondrial suspension. Cover tube with parafilm and invert to mix contents thoroughly.
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Add correct volume of mitochondrial suspension to first cuvette (based on the following table), then invert to mix thoroughly. Every 30 seconds, add mitochondrial suspension to the next cuvette. Do not add mitochondrial suspension to blanks.
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After 5 minutes, adjust spectrophotometer with blank #1 and take absorbance of tube 1. Adjust again with blank #2 and take absorbance of tube 2, then again with blank #3 for tubes 3 – 7.
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Take the absorbances for the seven tubes every 5 minutes for 35 minutes, always adjusting the spectrophotometer using the blanks for their corresponding tubes.