Isolation of Plasmid DNA: Separation on Size and Alkaline Denaturation (24 mins) | Lecture 47/90 | ASRB NET (ICAR-NET) Veterinary Microbiology

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Techniques in Microbiology and VaccinologyPrinciple of Molecular Techniques

Isolation of plasmid DNA

Separation on the basis of SIZE

Cell disruption carried out very gently to prevent wholesale break age of the bacterial DNA. For E. coli and related species, controlled lysis is performed.

For E. coli and related species, controlled lysis is performed as shown in Figure 3.11. Treatment with EDTA and lysozyme is carried out in the presence of sucrose, which prevents the cells from bursting immediately. Instead, spheroplasts are formed, cells with partially degraded cell walls that retain an intact cytoplasmic membrane.

Cell lysis is now induced by adding a non-ionic detergent such as Triton X-100(ionic detergents, such as SDS, cause chromosomal breakage). This method causes very little breakage of the bacterial DNA, so centrifugation leaves a cleared lysate, consisting almost entirely of plasmid DNA.

Separation on the basis of conformation

Alkaline denaturation

sodium hydroxide is added to a cell extract or cleared lysate, so that the pH is adjusted to 12.0 – 12.5, then the hydrogen bonding in non-supercoiled DNA molecules is broken, causing the double helix to unwind and the two polynucleotide chains to separate.

acid is now added, these denatured bacterial DNA strands reaggregate into a tangled mass. The insoluble network can be pelleted by centrifugation, leaving plasmid DNA in the supernatant.

Ethidium bromide – caesium chloride density gradient centrifugation

A density gradient is produced by centrifuging a solution of caesium chloride (CsCl) at a very high speed.

Macromolecules present in the CsCl solution when it is centrifuged form bands at distinct points in the gradient.

More importantly, density gradient centrifugation in the presence of ethidium bro mide (EtBr) can be used to separate supercoiled DNA from non-supercoiled molecules