Nuclei And Mitochondria Lab Report

Isolation of Nuclei and Mitochondria from Cauliflower Florets by Differential Centrifugation

Nuclei and mitochondria are both organelles that are found within most eukaryotic cells. The nucleus contains most of the genes needed for classification. It is "one of the most prominent structures to be encountered in the eukaryotic cell" (Schwarz 24). Nuclei were first observed by a Scottish plant taxonomist name Robert Brown in 1831. He studying Orchidaceae and Asclepiadaceae at the time when he noticed a structure in the cell that was consistent with much of the cells he was viewing. He termed this "the nucleus" (Enersen 1). Later in the 19th century scientists started using dyes to stain the nucleus. It was

Repetition of the centrifugation with more time and higher velocity can cause the release of the smaller, less dense particles.

In this experiment isolation of nuclei and mitochondria is completed by differential centrifugation. Once the organelles are separated they can be observed through a microscope. The materials needed to produce this experiment are: enough cauliflower to yield a 30g sample of floret tissue, extraction buffer (0.02M potassium phosphate, 0.3M D-mannitol, 0.4M sucrose, 2.0M magnesium chloride, and 0.02M glucose), grinding sand, cheesecloth, 50mL centrifuge tubes, small plastic capped culture tubes, a razorblade, a mortar and pestle, 70% ethanol-water solution, refrigerated centrifuge, microscope, microscope slides and cover slips, a spatula, Janus green stain, and aceto-orcein stain.

To perform this experiment one must first pre-chill the extraction buffer, mortar and pestle, test tubes, and any other instruments used to 3-5 degrees Celsius. Next, remove the outer 5mm of floret

Place it in the mortar and pestle that is already in the ice bucket. Add a pinch of grinding sand along with 15mL of extraction buffer and grind until the mixture becomes uniformly homogenized. Add another 15mLof extraction buffer and grind for an additional two minutes. Then, remove the grindate and pour it through four layers of cheesecloth that has been draped over a pre-chilled beaker. Wash the mortar with 5mL of extraction buffer to capture any excess into the cheesecloth. Squeeze the remaining liquid form the cheesecloth into a beaker. Afterwards, pour the extract into a 50mL centrifuge tube. Place the tube into the centrifuge making sure that it is balanced against another tube to avoid damage. Centrifuge for 10 minutes at 600xg and 4 degrees Celsius. Remove the tube and transfer the supernatant fluid into a cold 50mL centrifuge tube. Again, centrifuge the supernatant for 30 minutes at 20,000xg and 4 degrees Celsius. After the centrifugation completes, remove the tube and resuspend the mitochondrial pellet in 5mL of extraction buffer. Completely remove any clumped