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Chapter 13 : Restriction Analysis of Lambda DNA

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Abstract:

This chapter discusses the restriction analysis of lambda DNA. In the chapter, a series of steps are carried out that molecular biologists and biotechnologists use very frequently in their work. Restriction fragments of DNA are separated by electrophoresis through an agarose gel, the fragments are stained so that you can see them, and the products of the digest are analyzed. Predigested DNA samples are used or the digestions are set up and carried out. The process from restriction digestion to analysis of gel patterns is called “restriction analysis.” The procedure has been broken into several parts. The first part is setting up the restriction digests. The remaining steps are of preparing and loading the agarose gels, staining the gels, and analyzing the data. The DNA analyzed is from bacteriophage lambda. Lambda is a virus that infects and destroys it. Lambda DNA with the enzymes EcoRI, HindIII, and possibly BamHI is cut. These enzymes cut the DNA into a number of different-sized pieces called restriction fragments. The mixture of fragments is loaded into a well in the gel, and then electric current is applied. As DNA is negatively charged, the fragments will migrate toward the positive electrode in the gel box. The shorter the fragment, the faster it can progress through the agarose. The DNA is stained so that the fragments can be seen and results can be analyzed by comparing them to a restriction map of bacteriophage lambda.

Citation: Kreuzer H, Massey A. 2008. Restriction Analysis of Lambda DNA, p 194-198. In Molecular Biology and Biotechnology: A Guide for Students, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817480_ch13

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Agarose Gel Electrophoresis
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Figures

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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 of Lambda DNA, p 194-198. In Molecular Biology and Biotechnology: A Guide for Students, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817480_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 of Lambda DNA, p 194-198. In Molecular Biology and Biotechnology: A Guide for Students, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817480_ch13
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References

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Tables

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Table 13.1

Guide for setting up restriction digests

Citation: Kreuzer H, Massey A. 2008. Restriction Analysis of Lambda DNA, p 194-198. In Molecular Biology and Biotechnology: A Guide for Students, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817480_ch13
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Citation: Kreuzer H, Massey A. 2008. Restriction Analysis of Lambda DNA, p 194-198. In Molecular Biology and Biotechnology: A Guide for Students, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817480_ch13

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