DNA Isolation

Analysis of recombinant DNA assemblies often depends on the isolation of the plasmid DNA from the host cell. For this purpose, small scale preparation (mini-prep) of plasmid DNA is sufficient, and is most often performed by alkaline lysis. Plasmid DNA isolation by alkaline lysis begins by resuspending a cell pellet (typically from a saturated culture) in an isotonic solution followed by addition of an alkaline solution containing sodium dodecyl sulfate (SDS) to lyse the cells. During the lysis step, SDS disrupts the cell membrane and denatures proteins while the alkaline conditions denature the genomic DNA, plasmid DNA and proteins. After lysis, a neutralizing solution is added causing the denatured genomic DNA to precipitate, while the circular, covalently closed plasmid DNA reanneals, remaining in solution. The plasmid DNA is then separated from precipitated cellular debris and genomic DNA by centrifugation. Denatured protein contaminants are subsequently removed by phenol:chloroform extraction, followed by ethanol precipitation of the plasmid. Many commercially available kits use alkaline lysis followed by purification of plasmid DNA by ion-exchange resin, eliminating the need for phenol:cholorform clean up and ethanol precipitation. Other less frequently used methods of plasmid DNA isolation include equilibrium centrifugation with a cesium chloride gradient, and differential precipitation with polyethylene glycol.

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Protocols for DNA Isolation