Fractionation Isolation of Mitochondrial Fraction Tissue Preparation and Fractionation 1 Collect the mortar pestle and a styrofoam tray from your TA Fill the tray with ice and place the mortar and pestle in the ice 2 Use a razor blade to dice 20 g of cauliflower Each group will need 20 g of tissue 3 Place the tissue in a chilled mortar with 40 mL of ice cold Mannitol Grinding Buffer Grind the tissue vigorously with a chilled pestle for 4 min 4 Filter the suspension through four layers of cheesecloth into a chilled 50 mL centrifuge tube Wring out the liquid into the tube This liquid is known as the Filtrate F fraction Save 4 mL of Filtrate into a separate 15 mL falcon tube for later use Place on ice It is essential that all samples and materials be kept on ice at all times 5 Centrifuge the remaining filtrate F at 1000x gravity for 10 min at 0 4 C Make sure the centrifuge tubes are balanced and placed opposite one another in the centrifuge 6 Separate the pellet P1 from the supernatant S1 by pouring the supernatant into a clean chilled centrifuge tube 7 Set aside 4 mL of S1 into a 15 mL Falcon tube for later use Store on ice 8 Use a Pasteur pipette to resuspend P1 and scrape the mitochondrial pellet from the wall of the centrifuge tube and then with a Pasteur pipette thoroughly re suspend the sediment in 8 mL of ice cold Mannitol Assay Buffer Completely disrupt all of the chunks of cell debris Keep the fraction on ice 9 Centrifuge the remaining S1 at 12 000x gravity for 30 min at 4 C 10 During the centrifugation begin microscopy on F S1 and P1 You will create a wet mount for each sample F S1 and P1 using a slide and cover slip Use the instructions guide provided to complete the microscopy using the Zeiss Microscopes Axiocam and Axio Vision software Use the microscope to observe a drop from each of the first three fractions Filtrate S1 and P1 WHAT DO YOU SEE Are there whole cells present that were not disrupted during homogenization Use Azure C dye to stain nuclei by adding a drop of the dye to each slide from above Do you see any nuclei Describe the results in your notebook These images will be used in your report 11 After the centrifugation of S1 separate the supernatant S2 from the pellet P2 by pourin pernatant into a 50 mL falcon tube Save 4 mL of S2 for later use keep on ice