Biotech Basics: What happens if I Make the Wrong Percentage Agarose Gel?

Electrophoresis is a biotechnology technique can be used to separate dyes, proteins, and nucleic acid like DNA and RNA. Because of its ease of use and its ability to separate different molecules, electrophoresis has become one of the most common techniques used in the research laboratory.

Electrophoresis uses electricity and a porous gel matrix to separate mixtures of molecules into discrete zones, or bands, based on the physical properties of the molecule. This includes the molecule’s charge, its shape, and its size.

The structure of the repeating unit of an agarose polymer by Yikrazuul

One step that’s easy to overlook is the preparation of the agarose gel. Agarose is a long chain of simple sugar molecules, or polysaccharide, which is purified from seaweed.  When prepared correctly, the agarose acts like a strainer, or sieve, helping us to separate molecules by size. While a cast agarose gel looks and feels like a solid, at the molecular level it is full of tiny holes, or pores.  The pores influence the way that DNA travels through the gel.  As the current is pushing the DNA fragments through the gel, they must find their way through these pores.  Since it’s easier for small molecules to fit through the pores than larger molecules, the DNA separates into bands by size. 

The size of the pores in the gel change based on the percentage of agarose in the gel.  Higher percentage gels have smaller pores, and lower percentage gels have larger pores.  This means that larger fragments separate better in low percentage gels because they fit through the channels better, whereas smaller fragments separate better in high percent gels because the smaller channels slow their movement. Higher percentage agarose gels do take a bit longer to run, which makes sense because the “tunnels” through the gel are smaller.  But, if we had a mixture of small DNA fragments, we might choose to use a high percent gel to get better resolution between the bands. So, when choosing a gel percentage for our experiments, we balance resolution with speed. 

Each Edvotek kit specifies a specific percentage of agarose to use in our gel. But, what happens if we make and use the wrong percentage gel? We answer that question — through hands-on experimentation — in this FREE video available on our YouTube Channel. Be sure to like the video and subscribe for updates!