Practical - DNA Extraction
Step 1: Mechanical Disruption: Mash the biological sample (e.g., fruit) in a plastic bag or mortar. This physically breaks the cell walls and increases the surface area for chemical reagents to act upon.
Step 2: Lysis Buffer Addition: Mix the mashed sample with a solution of detergent and salt. The detergent dissolves the lipid membranes, while the salt begins the process of neutralizing the DNA's charge.
Step 3: Filtration: Pour the mixture through a coffee filter or gauze into a clean beaker. This step is crucial for removing large cellular debris, cell walls, and un-mashed tissue, leaving a clear 'filtrate' containing the DNA.
Step 4: Precipitation: Slowly pour ice-cold ethanol down the side of the container to form a layer on top of the filtrate. The DNA will appear as white, cloudy, stringy fibers at the junction where the two liquids meet.
| Component | Role in Extraction | Physical Effect |
|---|---|---|
| Detergent | Membrane disruption | Dissolves lipids to release DNA from nucleus |
| Salt () | Charge neutralization | Allows DNA strands to aggregate by shielding negative charges |
| Ethanol | Precipitation | Reduces DNA solubility, making it visible as a solid |
| Temperature | Preservation | Cold alcohol prevents thermal degradation and slows enzyme activity |
It is important to distinguish between the filtrate (the liquid that passes through the filter) and the precipitate (the solid DNA that appears after adding alcohol).
While mechanical mashing breaks cell walls, it is the chemical detergent that is required to break the internal nuclear envelope to access the genome.
Temperature Control: Always emphasize that the ethanol must be ice-cold. Warm ethanol may allow the DNA to remain partially soluble, significantly reducing the visible yield.
The 'Why' of Reagents: Be prepared to explain the specific function of each reagent. A common exam question asks why detergent is used; the answer must mention 'breaking down cell/nuclear membranes' or 'dissolving lipids'.
Observation Skills: DNA is described as 'white', 'stringy', or 'cloudy'. Avoid describing it as 'clear' or 'liquid' once it has precipitated.
Safety and Accuracy: Mention that mashing should be thorough but not so violent that it shears the long DNA strands into tiny fragments, which would make them harder to see.
Mixing Layers: A common mistake is shaking the tube after adding ethanol. The ethanol must remain as a distinct layer on top; mixing them too vigorously can cause the DNA to re-dissolve or become lost in the bulk solution.
Confusing Cell Walls and Membranes: Students often think mashing is enough to get DNA. Mashing only breaks the rigid cell wall; the detergent is strictly necessary to break the lipid-based plasma membrane and nuclear membrane.
Contamination: Using dirty equipment can introduce DNases (enzymes that eat DNA) from the environment or skin, which can degrade the sample before it is observed.