Question

Explain the isolation of genomic (chromosomal) DNA (from bacteria/plant cell/ animal cell, by cell lysis).

Answer 1

Genomic (chromosomal) DNA from either bacteria, plant or animal cell can be isolated by lysis method. For plant cell or bacterial cell, first cell wall is mechanically disrupted. The cell wall is broken enzymatically using enzymes like lysozyme. After this, the cell membrane is disrupted using detergents and surfactants. The next step is precipitation of DNA. This step is done in order to separate DNA from the other cellular debris. In this step, sodium acetate is used to neutralize the negative charges on the DNA molecules. This makes the DNA more stable and less water soluble. After this, ice-cold isopropanol is added to precipitate DNA from the aqueous solution. The next step is purification step. Once the genomic DNA is released in the solution, phenol-chloroform solution is used to purify the sample. Phenol denatures the protein. After centrifugation, denatured proteins stay in the organic phase and DNA stays in the aqueous phase. The chloroform removes phenol residues from solution. The alcohol is added to remove any unwanted material and cellular debris. The purified DNA is then dissolved in the TE buffer.