Plasmid isolation takes advantage of the unique structural properties of plasmids. Plasmids are small, supercoiled circular pieces of DNA. Unlike the much larger bacterial chromosome (which is also circular), plasmids are quite resistant to permanent denaturation. Today, most laboratories use commercial kits for plasmid isolations, because the kits are convenient and relatively inexpensive. The kits give good yields of high-quality DNA, while avoiding the need for organic denaturatants. A variety of less expensive, but somewhat more time-consuming, procedures have been described for investigators who want to make their own reagents. These latter procedures generally give good yields of DNA, but the DNA is often less pure than DNA isolated with kits. Whatever the isolation procedure, the general principles of plasmid isolation are the same.
The figure and paragraphs on the opposite page summarize the steps and general principles used for plasmid isolation.
- Lysis and denaturation - Strong denaturating conditions are used to weaken the tough bacterial cell wall. The most common procedures use a combination of strong base and a detergent. The detergents help to solubilize lipids in the cell wall, allowing the denaturants to enter the cell. Proteins, because of their fragile structures, are irreversibly denatured. The treatment also breaks the hydrogen bonds holding together the two strands of DNA helices.
- Neutralization - Neutralization allows complementary DNA strands to reanneal and causes proteins to precipitate. Plasmids renature because they have supercoiled structures that have held the two strands of the helix together during denaturation. Chromosomal DNA is not able to renature, however, because its longer strands have become mixed with denatured proteins. Samples must be mixed gently at this step to prevent fragmentation of the long, chromosomal DNA into pieces that might be able to reanneal and co-purify with the plasmids.
- Centrifugation - Plasmid DNA is separated from large aggregates of precipitated proteins and chromosomal DNA by centrifugation.
- Additional purification - Plasmids are further purified by organic extraction or adsorption to a resin.
The plasmids that we are working with in this lab have been maintained in a laboratory strain of Escherichia coli. E. coli is a proteobacterium that normally inhabits the intestinal tract of warm-blooded mammals. The virulent strains of E. coli that appear in the news have acquired, often by lateral gene transfer, pathogenicity islands containing genes for virulence factors, tox- ins and adhesion proteins important for tissue invasion. Pathogenicity islands are not present in lab strains, which are derivatives E. coli strain K12. K12 is a debilitated strain that is unable to colonize the human intestine. It has been propagated and used safely in the laboratory for over 70 years. The E. coli strains used in molecular biology have also been selected to tolerate large numbers of plasmids.
We will use the ZyppyTM (Zymo Research) kit to purify plasmids from the transformedE. coli strains. The final purification step in the procedure involves a spin column of silica resin. Nucleic acids bind tightly to silica in the presence of high concentrations of salt. Following a wash step that removes any residual proteins, the plasmids are eluted with a low salt solution. Plasmid solutions are very stable and can be stored for long periods of time in the refrigerator or freezer.