ISOLATION OF DNA FROM ONION
This project will demonstrate several basic steps required for isolation of chromosomal DNA from cells. To extract the chromosomal DNA, both the cell membrane and the nuclear membrane must be broken open. This is accomplished by disrupting the membranes with a solution of detergent and salt,creating a cell homogenate. Once the DNA is released from the nucleus, it must be protected from nucleases, enzymes which will degrade the DNA. Keeping the cell homogenate cold and various chemical components of the homogenization medium help restrict the action of these nucleases. The final step of this protocol involves precipitation of DNA from the homogenate. When the homogenization medium is added in the first step, the positive ions of the salts (sodium chloride and sodium citrate) bind to the negatively charged DNA backbone, creating a DNA molecule with a neutral charge. When a cold polar solvent, like ethanol, is added to the DNA solution, the DNA is precipitated out of solution, leaving other cell components behind (proteins, lipids, polysaccharides, general cell debris). DNA can be isolated and precipitated using tissue from an onion. The cells are disrupted by manually chopping the onion into small bits, adding homogenization medium to the onion bits (a solution of dish detergent and salts), and then grinding this mixture in a blender. The pureed onion is filtered with cheesecloth to remove any solid material, resulting in a clear cell homogenate. Ice cold ethanol is added to this homogenate to precipitate the DNA. The DNA can then be isolated by slowly stirring a glass stirring rod in the homogenate, literally spooling the DNA onto the glass rod.
The goal of this exercise is to demonstrate the isolation of DNA from living tissues. a. isolate DNA from onion tissue.
b. make observations regarding the results of the isolation.
c. develop a hypothesis.. about the nature of DNA molecule.