Isolating Plasmid DNA

Strategy

Bacteria actually do most of the work in plasmid DNA production. Bacteria are usually grown to stationary phase in liquid media so as to produce the maximum amount of plasmids per ml of culture. As the bacteria grow and divide they also replicate the plasmids that we force them to carry.

Plasmids are then isolated from the cultures. There are many protocols for isolating plasmid DNA from bacterial cells but they all contain the same two basic steps: lyse the cells and separate the plasmid DNA from the other cell components.

Usually cells are lysed either using heat, or using alkaline conditions.
Once the cells are lysed, the plasmid DNA is separated from the rest of the cellular components using a combination of chemical and physical techniques. This sounds a lot more complicated than it is.

Volume of bacterial cells needed

The amount of culture needed depends on the approximate yield of plasmid DNA per milliliter of culture.

Plasmid yield per ml is primarily dependent on the plasmid copy number (the average number of plasmids per cell). Plasmid copy number is controlled by a plasmid's replicon, which includes the DNA replication origin (the ori) and DNA encoded replication control elements. In plasmids, the ori and regulatory elements are usually found close together.

More than 30 different plasmid replicons have been described but almost all plasmids used in molecular cloning carry a replicon derived from pMB1 (Sambrook and Russell, 2001). The naturally occurring pMB1 replicon has a copy number of 15 to 20 plasmids per bacterial cell. However, vectors derived from pMB1, such as the pUC family or the pGEM family, carry highly modified replicons that can be maintained at much higher copy numbers. For example, the plasmid pKC7 is a pBR322 derivative that carries the un-altered pMB1 replicon and under normal conditions, is maintained at 15 to 20 copies per cell. On the other hand, pGEM-T ®, is maintained at 500 to 700 copies per cell.

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