Isolation of plasmid DNA by boiling lysis method and analysis by gel electrophoresis
The bacterial cell is enclosed in a cytoplasmic memberane and surrounded by a rigid cell wall with same species including E.coli the cell wall may itself be enveloped by a second outer memberane. All of these barriers have to be disrupted to release the cell components. The chemical used to break the cell wall are sodium chloride, Tris, EDTA and Triton X- 100 (STET). Tris is used to maintain the pH, EDTA removes magnesium ions that are essential for preserving the overall structure of the cell envelope. Triton X – 100 helps in the weakning of cell wall. Lysozyme is also used in this method. It is an enzyme that is present in egg, tears and saliva. Cell are then lysed by heating to 100ºC. In addition to cracking, heating disrupts base pairs of DNA strands and denatures the proteins and chromosomal DNA of the host. However, the strands of closed circular plasmid DNA are unable to separate from each other because their phosphodiester backbone are topologically interwined, when the temperature is lowered the bases of closed circular molecules of DNA falls once again into register forming super helical molecules. After denatured, chromosomal DNA and proteins have been removed by centrifugation, native plasmid DNA can be recovered from the supernatant.