Protocol for Lab 5 – Aerobic Respiration Part 1 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. 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 (urn) 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