plasmid

Plasmids are extra-chromosomal genetic elements present in most species of Archaea, Eukarya and Eubacteria that can replicate independently. Plasmids are circular double stranded DNA molecule that are distinct from the cells chromosomal DNA. The isolation of plasmid DNA from bacteria is a crucial technique in molecular biology and is an essential step in many procedures such as cloning, DNA sequencing, transfection, and gene therapy. Alkaline lysis is a method used in molecular biology, to isolate plasmid DNA or other cell components such as proteins by breaking the cells open. Bacteria containing the plasmid of interest is first grown, and then allowed to lyse with an alkaline lysis buffer consisting of a detergent sodium dodecyl sulfate (SDS) and a strong base sodium hydroxide. The detergent cleaves the phospholipid-bilayer of membrane and the alkali denatures the proteins which are involved in maintaining the structure of the cell membrane. Through a series of steps involving agitation, precipitation, centrifugation, and the removal of supernatant, cellular debris is removed and the plasmid is isolated and purified. 

Purification of plasmid DNA from bacterial DNA is based on the differential denaturation of chromosomal and plasmid DNA using alkaline lysis in order to separate the two. The basic steps of plasmid isolation are disruption of the cellular structure to create a lysate, separation of the plasmid from the chromosomal DNA, cell debris and other insoluble material. Bacteria are lysed with a lysis buffer solution containing sodium SDS and sodium hydroxide. During this step disruption of most cells is done, chromosomal as well as plasmid DNA are denatured and the resulting lysate is cleared by centrifugation, filtration or magnetic clearing. Subsequent neutralisation with potassium acetate allows only the covalently closed plasmid DNA to runnel and to stay solubilised. Most of the chromosomal DNA and proteins precipitate in a complex formed with potassium and SDS, which is removed by centrifugation.