The sample is mechanically broken (chopped or blended) and treated with a detergent solution to break down cell and nuclear membranes, releasing the DNA into the solution.
The mixture is heated to to facilitate membrane breakdown and denature DNase enzymes. It is then rapidly cooled in an ice bath to prevent the DNA itself from thermal degradation.
The mixture is filtered to remove large debris (cell walls, fibers). A protease enzyme is added to the filtrate to digest histones and other proteins that are tightly bound to the DNA strands.
Ice-cold ethanol is slowly poured down the side of the tube. Because DNA is insoluble in cold alcohol, it precipitates out of the aqueous solution as visible white, stringy fibers at the interface.
| Component | Primary Function | Mechanism of Action |
|---|---|---|
| Detergent | Membrane Disruption | Solubilizes lipids in the phospholipid bilayer. |
| Protease | Protein Removal | Hydrolyzes peptide bonds in histones and enzymes. |
| Ethanol | Precipitation | Reduces the dielectric constant, making DNA insoluble. |
| Heat () | Enzyme Inactivation | Denatures DNase to protect the target DNA. |
Temperature Control: Always emphasize that ethanol must be ice-cold. If the ethanol is warm, the DNA will remain soluble and fail to precipitate, resulting in a zero yield.
Blending Duration: Note that blending must be brief (e.g., 5-10 seconds). Excessive blending creates mechanical shear forces that can snap the long DNA polymers into small, unusable fragments.
Sequence of Reagents: Remember that protease must be added after filtration but before ethanol. Adding it after ethanol is useless because the proteins will already be trapped in the precipitate or the DNA will have already crashed out.
Misconception: Students often think the white precipitate is pure DNA. In reality, it is a complex of DNA and some residual salts or proteins, though it is 'purified' relative to the starting cell mass.
Error in Heating: Heating above for too long can cause the DNA double helix to denature (unzip), which may be undesirable for certain types of analysis.
Filter Choice: Using standard paper filters is necessary; using cloth or coarse mesh may allow too much cellular debris to pass through, contaminating the final precipitate.
