This protocol for DNA isolation is universal for most biological specimens. The method will effectively isolate DNA from whole blood, bones, plant samples, soil, herbarium, the mycelium of fungi, and tissues rich in secondary metabolites, polysaccharides, and pigments. DNA samples obtained using the proposed method can be used in studies where the presence of contaminants in nucleic acids is undesirable; for example, during cloning, sequencing, and genotyping.
All lab procedures are performed at room temperature. Prepare all solutions using ultrapure Milli-Q water and analytical grade reagents. Prepare and store all reagents at room temperature unless otherwise specified and away from direct sunlight.
The composition of the lysis solution contains inorganic salts (sodium chloride), within the effective concentration in the range of 1–4 M. The optimal concentration of the detergent is 1.5% CTAB. To increase the efficiency of DNA extraction, proteinase K can be added to the acidic lysing solution, which retains proteolytic activity at high ionic strength and low pH values, even in the presence of strong detergents and chaotropic agents. The subsequent extraction with chloroform increases the purity of the isolated DNA, especially from complex samples (thermally treated raw materials, blood, herbarium specimen, and soils). The effective concentration of chloroform is 1–2 volumes of the total lysate. Further, the DNA is precipitated from the aqueous phase with a water-soluble organic solvent, such as ethanol (it is preferable to use isopropanol). Depending on the biological material, DNA can be precipitated by filtration through a column with a glass microfiber filter, for example, Glass microfiber filters (Grade GF/A) or through cellulose paper. Finally, DNA is washed by precipitation or filtration in a solution of 80% ethanol and dissolved in low ionic buffered water.