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Isolation of Mitochondria from Cauliflower

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Isolation of Mitochondria from Cauliflower – Weigh egg of rosettes cut from fresh cauliflower head. – Cut rosettes and place it on ice – Prepare Juice extractor by placing ice and an empty 150 ml beaker into the right compartment. – Collect pulp from left compartment and record total volume of the extract. Approve. Ml – Filter the pulp using six layered cheese cloth and collect it in a beaker sitting on ice. – Place two 50 ml test tubes in the ice bucket A and B.

Add 10 ml of cold isolation buffer and 10 ml of extract to tube A.

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Mix thoroughly and centrifuge at 3000 RPM for 10 min. (Make sure tubes are balanced before running centrifuge. Weight- balance other tube with water to make it equal to tube A). – Collect supernatant and add to tube B. – Pellet in tube A contains cell debris, cells, cell wall material, nuclei and mitochondria. Add Mil buffer to pellet and mix. – Prepare slide, place 1 drop of suspension plus methyl blue and place cover slip on it.

Centrifuge tube B at 2000 RPM for 30 min to get mitochondrial pellet and a supernatant. (Balance test tubes before centrifugation). – Respond the pellet (to avoid clumps) using cold stirring glass rod. Add one drop of assay buffer. Continue breaking clumps and adding assay buffer until ml buffer is added. – Mix the suspension and transfer to a precook 15 ml test tube M. – Prepare slide using suspension from M tube and adding Janis Green B dye to it. Cover using cover slip. – Add 1 ml of mitochondrial suspension too 15 ml test tube P.

Set spectrophotometer at Mann and measure absorbency. – Draw the standard curve for DIP on a linear graph paper. Draw best fit line. Succinctness Dehydrogenate Assay – Label four (13×100) test tubes 1 to 4. – Follow below table and add content to each tube. Buffer (ml) Aside (ml) Succinctness (ml) DIP (run) Mitochondria (ml) 1 blank 3. 5 part 3 3. 0 3 blank – Tubes 1 and 3 are blank. Read absorbency for tube 2 every 2 min for 14 min. Prepare a linear plot Absorbency versus time for tube 1 and 2, and 3 and 4

Cite this Isolation of Mitochondria from Cauliflower

Isolation of Mitochondria from Cauliflower. (2017, Oct 19). Retrieved from https://graduateway.com/biology-lab-report-6/

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