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Chapter 13 : Restriction Analysis

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Restriction Analysis, Page 1 of 2

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Abstract:

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. 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.

Citation: Kreuzer H, Massey A. 2008. Restriction Analysis, p 230-243. In Molecular Biology and Biotechnology: A Guide for Teachers, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816100.ch13

Key Concept Ranking

Agarose Gel Electrophoresis
0.53075594
Sodium Dodecyl Sulfate
0.48344073
DNA Restriction Enzymes
0.44039738
0.53075594
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Figures

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Ideal gel (the 125-bp HindIII fragment will not be visible; the 564-bp HindIII fragment probably will not be visible and is not shown).

Citation: Kreuzer H, Massey A. 2008. Restriction Analysis, p 230-243. In Molecular Biology and Biotechnology: A Guide for Teachers, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816100.ch13
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Image of

Ideal gel with fragment sizes in base pairs.

Citation: Kreuzer H, Massey A. 2008. Restriction Analysis, p 230-243. In Molecular Biology and Biotechnology: A Guide for Teachers, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816100.ch13
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Image of Figure 13.1
Figure 13.1

Bacteriophage lambda is the best known of the phages. It has a linear chromosome of approximately 48,500 bp and about 45 known genes. Its restriction map is shown.

Citation: Kreuzer H, Massey A. 2008. Restriction Analysis, p 230-243. In Molecular Biology and Biotechnology: A Guide for Teachers, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816100.ch13
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Image of Figure 13.2
Figure 13.2

Ideal gel pattern. The 125- bp HindIII fragment will never be seen. The 564-bp HindIII fragment is also usually not visible and is not shown in this example.

Citation: Kreuzer H, Massey A. 2008. Restriction Analysis, p 230-243. In Molecular Biology and Biotechnology: A Guide for Teachers, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816100.ch13
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References

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Tables

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Citation: Kreuzer H, Massey A. 2008. Restriction Analysis, p 230-243. In Molecular Biology and Biotechnology: A Guide for Teachers, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816100.ch13
Generic image for table
Table 13.1

Guide for setting up restriction digests

Citation: Kreuzer H, Massey A. 2008. Restriction Analysis, p 230-243. In Molecular Biology and Biotechnology: A Guide for Teachers, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816100.ch13
Generic image for table
Untitled

Citation: Kreuzer H, Massey A. 2008. Restriction Analysis, p 230-243. In Molecular Biology and Biotechnology: A Guide for Teachers, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816100.ch13

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