Alkaline lysis
Alkaline lysis is a method used in molecular biology to isolate plasmid DNA from bacteria.
Method
Bacteria containing the plasmid of interest are first cultured, then a sample is centrifuged in order to concentrate cellular material (including DNA) into a pellet at the bottom of the containing vessel. The supernatant is discarded, and the pellet is then re-suspended in an EDTA-containing physiological buffer. The purpose of the EDTA is to chelate divalent metal cations such as Mg2+ and Ca2+, which are required for the function of DNA degrading enzymes (DNAses) and also serve to stabilise the DNA phosphate backbone.
Separately, a strong alkaline solution consisting of the detergent sodium dodecyl sulfate (C12H25SO4Na) and a strong base such as sodium hydroxide (NaOH) is prepared and then added. The resulting mixture is incubated for a few minutes. During this time, the detergent disrupts cell membranes and allows the alkali to contact and denature both chromosomal and plasmid DNA.
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