Plasmid DNA Purification in Bacteria
Lab #4: Purification of Plasmid DNA
Introduction
Plasmids are circular DNA molecules that constitute accessory genetic material and replicate independently of the host cell chromosome. They are naturally found in bacteria, with sizes ranging from approximately 5,000 to 400,000 base pairs (bp).
The information plasmids contain may confer upon their host cell traits such as antibiotic resistance, the capacity for degradation of aromatic compounds, or the fermentation of sugars, among others. The number of plasmids present in a cell can vary; some bacteria do not contain any plasmids, while others contain several copies. If a plasmid has the ability to insert itself into the bacterial chromosome, it is called an episome.
Many methods have been developed to purify plasmid DNA from bacteria. All these purification methods invariably involve the following steps:
- Growth of bacterial culture.
- Collection and lysis of bacteria.
- Plasmid DNA purification.
Growth of Bacterial Cultures
It is possible to purify plasmids from a bacterial culture that has been inoculated from a single transformed colony taken from an agar plate. Usually, the colony is transferred to a small-volume liquid culture, which is grown to the final phase of logarithmic growth. This culture can be used to prepare small amounts of plasmid DNA (minipreparation) or to inoculate larger-scale cultures that will be used to prepare larger amounts of plasmid DNA (midi and maxipreparations). All the while, the transformed bacteria can be grown selectively using appropriate antibiotics.
Collection and Lysis of Bacteria
Bacteria are harvested by centrifugation from a bacterial culture and are then lysed by one or more methods, including ionic and nonionic detergents, organic solvents, alkali, and heat. The choice of lysis method is determined by three factors: the size of the plasmid, the strain of E. coli, and the technique subsequently used for plasmid purification.
Plasmid DNA Purification
The methods used allow for obtaining plasmid DNA that is always contaminated with large amounts of RNA and chromosomal DNA from E. coli. However, these contaminants must be removed or reduced to minimum levels to obtain good-quality plasmid DNA that can be used, for example, in mammalian cell transfection. Once purified, the plasmid DNA can be visualized in agarose gels and can be used as a substrate for many restriction enzymes and DNA polymerases.
The alkaline lysis method, in combination with the detergent SDS, has been used for more than 20 years to isolate plasmid DNA from E. coli bacteria (Birnboim and Doly, 1979). Exposure of bacteria to an ionic detergent and high pH opens the cell wall, denatures chromosomal DNA and proteins, and releases plasmid DNA into the supernatant. Although the alkaline solution breaks the paired bases, the circular plasmid DNA strands are unable to separate from each other and remain topologically intertwined. Extended exposure to OH– is harmful because plasmid DNA strands are not able to reconstitute when the pH returns to neutral.
During lysis, proteins, bacterial cell walls, and broken chromosomal DNA complexes become surrounded by sodium dodecyl sulfate (SDS). These complexes are efficiently precipitated when the solution’s sodium ions are replaced by potassium ions (Ish-Horowicz and Burke, 1981). The denatured material is then removed by centrifugation. Circular plasmid DNA can be recovered from the supernatant in different ways, depending on the subsequent use of the plasmid DNA. Alkaline lysis in the presence of SDS is a flexible technique for working with all strains of E. coli, and it also enables working with culture volumes in the range of 1-500 ml.