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Cell Fractionation and DNA Isolation

Description

Students isolate nuclei from calf thymus tissue and examine them microscopically. The DNA is then extracted from the nuclei by a simple procedure that uses a detergent and alcohol. Microscopes and a small centrifuge are desirable but not absolutely necessary for the exercise.

 

Sample

A sample from the student manual:

Background Information
A. The Eukaryotic Cell
Cells are frequently classified into two basic types: prokaryotic and eukaryotic. The prokaryotic cell, so named because it lacks a discrete nucleus, is found in bacteria and blue-green algae. Prokaryotic cells have relatively simple internal structures, although their metabolic activities are as complex as those in humans. The eukarytoic plan is observed in true algae, fungi, protozoans, and the cells of higher animals and plants. The eukaryotic cell, by definition, has a nucleus that contains most of the cell’s DNA. The nucleus is enclosed by a double membrane, the nuclear envelope, that separates nuclear components from the cytoplasm. The eukaryotic cell also contains a number of other specialized internal structures not found in prokaryotes including mitochondria, chloroplasts, and a rich array of internal fibers and membranes. A diagram of a typical animal cell is given in Figure 2-1.

The genetic material in a prokaryotic cell usually consists of a single circular molecule of DNA that is compacted within the cell interior. In contrast, DNA in the eukaryotic nucleus is partitioned into a number of chromosomes, and the DNA of an entire chromosome is thought to consist of a single linear molecule. Human cells contain 46 chromosomes and the average extended length of each chromosomal DNA molecule is about 4 centimeters. Therefore, the length of DNA molecules in length of DNA in an individual composed of 1014 cells is 2×1014 meters. This length is about 200 times greater than the distance from the earth to the sun! Clearly the DNA must be condensed or folded in some manner to fit within the confines of the nucleus.
In the nucleus of a typical eukaryotic cell, individual chromosomes can be identified only during cell division. However, a eukarytoic cell spends most of the life cycle in interphase, which is the stage between cell divisions. In interphase, the chromosomes are diffuse and the chromosomal material is called chromatin. In addition to DNA, chromatin and chromosomes contain proteins called histones, which are present in essentially all cell types in both plants and animals. Histones are basic in nature because they have large amounts of the basic amino acids arginine and lysine. These basic proteins associate with the acidic DNA backbone, folding the long DNA molecules into chromatin and compact chromo- somes. When these proteins are dissociated (removed) from chromatin, the DNA molecules unfold, resulting in an increase in the viscosity of the solution.

Student Manual

B1-2 Student Manual