Chromosomal DNA Purification

Extraction of chromosomal DNA from eukaryotic cells is the common procedure in molecular biology experiments and forensic analysis. Chromosomes of eukaryotic cells are located within the nucleus of the cell. Each eukaryotic chromosome consists of a single DNA molecule wrapped around the histone proteins. In order to get the chromosomal DNA out of the cell following steps are used.

1) Cells are treated with detergents such SDS (Sodium Dodecyl Sulfate), Triton-X-100 or Tween in order to break open the cell. These detergents will break the plasmamembranes and nuclear membrane of the cell and cytoplasmic material & proteins are digested. (The membranes are dissolved using detergents that solubilize the phospholipids that make up these membranes).

2) Once the membranes are dissolved, with the help of centrifuge, the cell lysate is formed. This cell lysate contains the cytoplasmic material, organelles and nuclear material and chromosomes.

3) The proteins are then treated with the enzyme called Proteases such as Protease K which will digest these proteins.

4) After the protease treatment, the DNA will be present in Aqueous Solution.

5)Then the cell lysate is treated with the mixture of phenol and chloroform which are organic solvents. This mixture will remove the proteins and other cell debris. They leave the DNA in the aqueous interphase between phenol and chloroform.

6) The above step is repeated to further purify the DNA. (When absolute 100% ethanol is layered on top of the aqueous DNA solution, the interphase develops between two liquids due to their difference in densities).

7) DNA in the aqueous solution (interpase) is then spooled using the glass rod or pipette. This pipetted solution is then transferred to fresh and clean tube. where the DNA can be dried and stored or can be diluted with aqueous solution and stored.

8) This purified DNA is then used for further experiments such PCR, gene analysis, cloning or molecular biology experiments.

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