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Chapter 13 : Restriction Analysis
In the activity described in this chapter, students perform electrophoresis using precut samples of lambda DNA, or they first carry out the restriction digests themselves and then perform electrophoresis with their samples. DNA Scissors include using restriction maps to predict the sizes of DNA fragments after digestion. The restriction map of bacteriophage lambda should be shown to them; so that they can predict the sizes of the fragments they will see in their gels. The standard method for separating DNA fragments is electrophoresis through agarose gels. Agarose is a polysaccharide, like agar or pectin that dissolves in boiling water and then gels as it cools. In the introductory material, this chapter provides information about different methods of staining DNA and recording data. The procedure outlined in the student activity was designed for the safest DNA stains, which are also the least sensitive. Methylene blue is an alternative stain for DNA gels recommended for classroom use by the National Association of Biology Teachers. The other gel material used in electrophoresis of DNA is polyacrylamide. Polyacrylamide forms a tighter mesh than does agarose, so polyacrylamide gels can separate smaller molecules. Proteins are normally separated by polyacrylamide gel electrophoresis, because proteins are much smaller molecules than the DNA fragments commonly separated on agarose. Protein electrophoresis offers several advantages over DNA electrophoresis. Students typically want to analyze DNA from familiar organisms, often seeking to identify organisms from restriction fragment patterns.