4-1.3.3.1.1. Separation based on size difference

•  It involves lysis of cells with lysozyme and EDTA (function as described above in point 4-1.2.2. ) in the presence of sucrose (prevents the immediate bursting of cell).

•  Cells with partially degraded cell walls are formed that retain an intact cytoplasmic membrane called as sphaeroplasts.

•  Cell lysis is then induced by the addition of a non-ionic detergent (e.g. Triton X-100) or ionic detergents (e.g.SDS) causing chromosomal breakage.

•  Bacterial chromosome attached to cell membrane, upon lysis gets removed with the cell debris.

•  A cleared lysate consisting almost entirely of plasmid DNA is formed with very little breakage of the bacterial DNA. (Figure 4-1.3.3.1.1.).

 

Figure 4-1.3.3.1.1: Separation of plasmid DNA on the basis of size.