Alkaline lysis

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Alkaline lysis is a method used in molecular biology to isolate plasmid DNA from relatively small volumes of bacteria.

Method[edit]

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 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 left on ice for a few minutes. During this time, the detergent disrupts the cells' membranes and allows the alkali into contact with both the chromosomal and plasmid DNA, which it denatures.

Finally, potassium acetate is added. This acidifies the solution and allows the renaturing of plasmid DNA, but not chromosomal DNA, which is precipitated out of solution. Another function of the potassium is to cause the precipitation of potassium dodecyl sulfate and thus removal of the detergent. A final centrifugation is carried out, and this time the pellet contains only debris and can be discarded. The plasmid-containing supernatant is carefully removed and used in further analysis, such as gel electrophoresis.

Other uses[edit]

Also, alkaline lysis is sometimes used to extract plant genetic material. The plant cells are subjected to a strongly alkaline solution containing a detergent (usually a zwitterionic or nonionic detergent such as Tween 20), and the mixture is incubated at high temperature. This method is not used as often due to the sodium hydroxide's tendency to damage genetic material, reducing DNA fragment size.

References[edit]