Alkaline Lysis Prep of GFP Plasmid from E. coli
Introduction
The ability to isolate plasmid DNA is crucial to recombinant DNA research and many other applications in molecular biology. Large-scale plasmid purifications often require time-consuming procedures such as cesium chloride density gradient centrifugation, gel filtration columns, or affinity columns. These techniques can produce highly purified plasmid DNA, but consume consider amounts of time and materials. However, not all manipulations involving plasmids require highly purified material. For example, when screening a large number of clones, it is more important to be able to rapidly isolate small amounts of relatively pure plasmid than to spend the time to prepare highly purified DNA. Several rapid small-scale plasmid isolation procedures have evolved and are widely used procedures in molecular biology. Because small amounts of bacterial cells are used and small amounts of plasmid are isolated, the procedures are referred to as mini-preps. Despite the small quantities of cells used, the yield of plasmid is quite impressive—usually in the range of 1 to 5 μg.
Plasmid purification procedures selectively enrich plasmid DNA over chromosomal DNA, which is present in the cell in much greater quantities. The two most widely used mini-preps are the alkaline lysis (Birnboim and Doly, 1979) and rapid boiling (Holmes and Quigley, 1981) procedures. Today we will use the alkaline lysis procedure. Both protocols involve a precipitation of cell debris and protein and take advantage of physical properties of plasmids to separate them from the chromosomal DNA. Either of these procedures can be done in 1 to 2 hours, allowing rapid screening of many different plasmids. The plasmid DNA produced in these protocols is relatively clean and can be used in restriction digestion, transformations, PCR, and even DNA sequencing.
In both procedures, cells are pelleted rapidly in a microcentrifuge and the pellet is resuspended in a buffered medium. In the alkaline lysis procedure, cells are lysed with a solution of SDS (sodium dodecyl sulfate) and NaOH. In addition to lysing the cells, these components serve several other valuable functions. The SDS and NaOH solubilize and denature cellular constituents, and the elevated pH will begin to degrade RNA. The alkaline conditions also result in DNA denaturation (strand separation). Because the chromosomal DNA is very large, it is broken by shear forces when the cells lyse, while the small plasmids remain intact. Thus, when denatured, the plasmids remain linked to their complementary strand—much as two links of a chain are held together. When the lysate is neutralized with potassium acetate, the hydrogen bonds reform. Because the complementary plasmid DNA strands are held in close proximity to each other by the linked strands, they will immediately find their complementary strand and base pair properly to form an intact double-stranded plasmid. However, the chromosomal DNA will not be near its complementary strand. When the solution is neutralized, the DNA will base pair, but not to its complementary strand. The result will be a rather large, interlinked mass of DNA strands. The elevated salt concentration resulting from the potassium acetate addition will cause the SDS and protein to form a flocculent precipitate.