Agarose gel electrophoresis is a precise analytical procedure that is widely used in biomedical, biotechnology, forensic, and research laboratories. It is the easiest way of separating, visualizing and analyzing DNA fragments and also separating other charges biomolecules such as dyes, RNA and proteins.
The usefulness of agarose gel for the electrophoresis of DNA and large protein molecules is based on the fact that it has large pore size and good gel strength. Agarose gels are typically cast and run using a TAE or TBE buffer. DNA molecules are moved using an electric field. Shorter molecules move more easily and faster across an agarose gel than longer molecules, a process called sieving.
The key to efficient electrophoresis is achieving higher resolution in agarose gels. According to experts, this depends on various factors such as
- Using the right buffer (TAE or TBE): The type of buffer chosen is based on the size of DNA fragments and the post-electrophoresis application. TBE buffer is ideal for small DNA (<1000 bp).
- Volume of DNA to be loaded for the best resolution: It is important to determine the quantity of DNA in the band or bands that is to be separated. The amount of DNA can be varied and the minimal quantity of DNA that can be detected depends on the stains used. Typically, loading DNA in the smallest volume possible will provide higher resolution (sharper bands).
- Thickness of the agarose gel: If the consistency of the agarose gel is too thick, it will result in fuzzy bands and higher staining background. Also if the quantity of the buffer is too high, it will reduce DNA mobility and result in band distortion. The thickness of the comb also affects resolution. A clean, thin comb results in well-defined bands.
- Running conditions: The voltage in the gel electrophoresis unit must be optimal – neither too high nor too low. If it is too high, the gel gets overheated and the band resolution is reduced. If the voltage is too low, it affects DNA mobility and diffusion causes the bands to broaden.
Finally, the type of gel used is crucial. Based on DNA application and size, the type of agarose used would affect DNA resolution. Consider gel strength and melting temperature during agarose selection. Compared to other products in the market, Benchmark Scientific’s Agarose LE is a good choice. It has a low EEO (≤ 0.13-mr) which promotes increased electrophoretic mobility, allowing the macromolecules and larger particles to migrate more freely through the gel matrix. It yields improved resolution and shorter run times, and the consistently low EEO ensures reduction in band distortion.