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How is a plasmid purified from a bacterial cell without contaminating genomic DNA?

Genomic DNA is present in a bacterium as a linear molecule usually a few megabases in size (~4Mb for Escherichia coli), while plasmids are closed circular DNA that are significantly smaller (2–200kb). Early efforts at isolating plasmid DNA free of genomic DNA were based on differences in DNA size and conformation and involved ultracentrifugation through cesium chloride gradients (1). Modern methods are based on the genome's attachment to the cell membrane versus free-floating circular DNA. After the cell is lysed, usually by SDS-alkaline denaturation (2,3), the genomic DNA is trapped with denatured proteins, which are pelleted during centrifugation. The resulting supernatant, which contains the circular plasmid, is removed and processed over silica membrane columns [e.g., PureYield™ Plasmid Miniprep System (Cat.# A1221)] or precipitated to purify and concentrate the plasmid DNA. In this manner, the plasmid DNA is purified and the genomic DNA removed. Regardless of the method used, it is important not to overload the purification system. Using too many bacterial cells can lead to genomic DNA copurification with the desired plasmid.

References

1. Clewell, D.B. and Helinski, D.R. (1969) Supercoiled circular DNA-protein complex in Escherichia coli: Purification and induced conversion to an open circular DNA form. Proc. Natl. Acad. Sci. USA 62, 1159–66.
2. Birnboim, H.C. and Doly, J. (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7, 1513–23.
3. Birnboim, H.C. (1983) A rapid alkaline extraction method for the isolation of plasmid DNA. Methods Enzymol. 100, 243–55.