Chapter 30 : Plasmids

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The elimination (curing) of a genetic trait by treating the bacterial population with chemical or physical curing agents such as acridine dyes, ethidium bromide, sodium dodecyl sulfate (SDS), antibiotics, high temperature, or electroporation indicates that the expression of that genetic trait is linked to the presence of a plasmid. This chapter deals with the isolation, purification, and characterization of bacterial plasmids and also provides some information concerning the novel application of plasmid biology in gene therapy. The chapter provides a representative rather than comprehensive account of techniques and references concerning the biology of plasmids and their use as tools in molecular biology and pharmacology. The lysostaphin and lysozyme method was adapted in large scale isolation methods. The cold alkaline pH method is a scaled-down version of the one described in large scale that has been used to prepare and plasmids. The acid phenol method is based on the addition of an acid phenol extraction to the cold alkaline lysis preparation of DNA and has been used very successfully. The boiling method described here is the original method of Holmes and Quigley, used effectively for screening of high-copy-number plasmids. The protocols to isolate linear plasmids involve cell lysis, in some cases followed by CsCl-ethidium chloride gradient purification, and the separation of the linear plasmid from the chromosomal DNA by either sucrose gradient or agarose gel electrophoresis.

Citation: Tolmasky M, Actis L, Welch T, Crosa J. 2007. Plasmids, p 709-734. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch30
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Determination of plasmid copy number. A plasmid-containing bacterial strain was grown in a minimal salts medium plus Casamino Acids and glucose. Labeling and lysis were carried out, and the lysate was centrifuged in the CsClethidium bromide gradient. Seven-drop fractions were collected on microtiter trays and assayed for radioactivity.

Citation: Tolmasky M, Actis L, Welch T, Crosa J. 2007. Plasmids, p 709-734. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch30
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Composition of commonly used reagents

Citation: Tolmasky M, Actis L, Welch T, Crosa J. 2007. Plasmids, p 709-734. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch30
Generic image for table

Determination of homology between heterologous plasmids by use of single-strand-specific endonuclease

The count-per-minute values were obtained by subtracting a background of 20 cpm. +, positive strand; ‒, negative strand.

Values in this column were obtained by subtracting the value in sample 2 from the respective values in samples 3, 4, and 5 and then normalizing to the corrected value in sample 3.

Citation: Tolmasky M, Actis L, Welch T, Crosa J. 2007. Plasmids, p 709-734. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch30

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