Agarose gel electrophoresis is a widely used laboratory technique for the separation and analysis of DNA fragments. This method exploits the negative charge of DNA, which allows it to migrate through a gel matrix under the influence of an electric field. The technique not only enables the visualization of DNA but also allows researchers to determine the approximate size of DNA fragments and assess the success of molecular biology experiments like PCR, cloning, or restriction digestion.
The process involves several critical steps, each of which requires attention to detail. Below is a detailed guide to each stage of agarose gel electrophoresis, along with helpful troubleshooting tips.
1. Preparing the Agarose Gel
The first step is to prepare the agarose gel, which acts as a molecular sieve through which DNA migrates. Agarose is a polysaccharide extracted from seaweed, and when dissolved in buffer and cooled, it forms a semi-solid gel. The concentration of agarose directly affects the resolution of DNA fragments of different sizes.
A lower percentage gel (e.g., 0.7%) is more porous and better suited for separating large DNA fragments.
Higher percentage gels (e.g., 2.0%) are ideal for resolving smaller DNA fragments below 200 base pairs.
Percentage of Agarose Gel for Resolution of DNA Fragments
% (w/v) Agarose
Optimum Resolution (kb)
0.5
1–30
0.7
0.8–12
1.0
0.5–10
1.2
0.4–7
1.5
0.2–3
2.0
0.05–2
A specific amount of agarose is weighed and added to an appropriate volume of electrophoresis buffer. Common buffers include:
TAE (Tris-acetate-EDTA)
TBE (Tris-borate-EDTA)
TPE (Tris-phosphate-EDTA)
While TAE is commonly used and suitable for DNA recovery and downstream applications, TBE provides sharper resolution for smaller fragments and is preferred for longer runs.
Once mixed, the solution is heated, usually in a microwave, until the agarose is completely dissolved. The solution must be clear, with no visible particles remaining. Overheating should be avoided, as it can alter the buffer composition and degrade agarose.
After heating, the solution is allowed to cool to approximately 55°C. At this stage, a DNA-binding dye such as ethidium bromide (EtBr) or a safer alternative like GelRed or SYBR Safe can be added. These dyes intercalate into the DNA and fluoresce under UV or blue light, making DNA visible after electrophoresis.
Once the dye is mixed, the solution is poured into a casting tray fitted with a comb to create wells for sample loading. Care should be taken to pour slowly and avoid air bubbles, which can interfere with DNA migration.
The gel is left to set at room temperature for 20–30 minutes, during which it solidifies into a firm yet flexible matrix.
Casting tray, comb, and gel tank. Image: Understanding PCR – A Practical Bench-Top Guide
2. Loading the DNA Samples
Once the gel is solidified, it is placed into the electrophoresis chamber, and the same buffer used in gel preparation is poured into the tank until the gel is completely submerged. The comb is then carefully removed, revealing wells.
Before loading the samples, DNA is mixed with a loading dye, which:
Increases the density of the DNA sample, allowing it to sink into the well
Adds color to make sample loading visible
Typically contains sucrose, glycerol, or Ficoll to increase density
Includes tracking dyes such as bromophenol blue or xylene cyanol to monitor migration
Loading the DNA into the wells requires a steady hand and fine pipetting technique. It’s helpful to use a black background under the tank to better see the wells.
A molecular weight ladder (DNA marker) must also be loaded into one of the wells, usually the first or last lane, as a reference for determining the size of DNA fragments in the sample.
Common Mistake: Accidentally puncturing the bottom of the well with the pipette tip, causing the sample to leak into the buffer, which results in a smeared or absent band. Beginners may find it helpful to practice loading water before using actual DNA.
Loading of DNA samples after mixing with the tracking dye. Image: Understanding PCR – A Practical Bench-Top Guide
3. Running the Gel
With the samples loaded, the lid of the electrophoresis chamber is closed and connected to a power supply. It is vital to orient the gel correctly, DNA is negatively charged and should migrate toward the positive (red) electrode (DNA runs toward red).
The voltage applied depends on the size of the gel and the desired resolution. Typically:
A voltage of 4–10 volts per centimeter of distance between the electrodes is used.
Usually, 5 V/cm is optimal.
Running the gel too quickly (i.e., at a high voltage) can cause the gel to overheat, distort the bands, or even melt the gel.
As the run progresses, the tracking dye visibly migrates through the gel, providing an indication of progress. Electrophoresis is usually stopped when the dye front has migrated two-thirds of the gel length, at which point the DNA fragments should be sufficiently separated.
4. Staining the Gel (If Not Pre-Stained)
If the DNA stain was not included in the gel, staining must be performed after the run. The gel is gently transferred to a staining tray and submerged in a solution of the DNA-binding dye.
Ethidium bromide is traditionally used.
Safer alternatives include SYBR Safe or GelGreen.
The staining process usually takes about 30–45 minutes. After staining, the gel is rinsed in distilled water for 10–15 minutes to remove excess dye, which can reduce background fluorescence during visualization.
Safety Note: Always use appropriate PPE when handling EtBr and dispose of all waste according to biosafety regulations.
5. Visualizing the DNA
Once stained, the gel is placed on a transilluminator for visualization:
If EtBr was used, a UV transilluminator is required.
Safer stains often fluoresce under blue light.
The DNA fragments appear as distinct bands, each representing fragments of a particular size. The molecular weight marker provides a scale to estimate the size of the bands in the samples.
For documentation, photograph the gel using a gel imaging system to create a permanent record.
If only faint bands are seen or none at all, potential issues may include:
Degraded DNA
Improper loading
Too low DNA concentration
Presence of primer dimers (seen as small bands near the bottom of the gel)
6. Interpreting the Results
The final step is interpreting the gel image. The position of the DNA band is compared to the molecular weight marker to estimate its size.
A single, sharp band at the expected size suggests success.
Multiple bands may suggest nonspecific amplification, contamination, or partial digestion.
If the observed band size matches the expected size, the experiment likely worked. For absolute confirmation, sequencing the product is recommended.
Conclusion
Agarose gel electrophoresis is a powerful, accessible, and versatile method for analyzing DNA. Mastering this technique requires understanding not only the steps involved but also the subtle factors that affect the outcome, from gel concentration and buffer choice to staining methods and electrophoresis conditions.
One response
Great content with good explanation