Plasmid preparation

A plasmid preparation is a method of DNA extraction and purification for plasmid DNA. Many methods have been developed to purify plasmid DNA from bacteria. These methods invariably involve three steps:

Plasmids are almost always purified from liquid bacteria cultures, usually E. coli, which have been transformed and isolated. Virtually all plasmid vectors in common use encode one or more antibiotic resistance genes as a selectable marker, for example a gene encoding ampicillin or kanamycin resistance, which allows bacteria that have been successfully transformed to multiply uninhibited. Bacteria that have not taken up the plasmid vector are assumed to lack the resistance gene, and thus only colonies representing successful transformations are expected to grow. Bacteria are grown under favourable conditions.

When bacteria are lysed under alkaline conditions (pH 12.0-12.5) both chromosomal DNA and protein are denatured; the plasmid DNA however, remains stable. Some scientists reduce the concentration of NaOH used to 0.1M in order to reduce the occurrence of ssDNA. After the addition of acetate-containing neutralization buffer the large and less supercoiled chromosomal DNA and proteins precipitate, but the small bacterial DNA plasmids stay in solution.

Kits are available from varying manufacturers to purify plasmid DNA, which are named by size of bacterial culture and corresponding plasmid yield. In increasing order, these are the miniprep, midiprep, maxiprep, megaprep, and gigaprep. The plasmid DNA yield will vary depending on the plasmid copy number, type and size, the bacterial strain, the growth conditions, and the kit.

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