Factors affecting migration of Nucleic Acids in Agarose Gel Electrophoresis

Agarose gel electrophoresis is a molecular biology technique that can be used to separate DNA, RNA, or protein. Below are the seven different factors that affect the movement of nucleic acids during electrophoresis:

Size of the DNA: This one is obvious, the bigger the DNA, the slower it will move while the smaller ones tend to move faster as can be seen in the following figure where the 100 bp DNA band runs faster than the 1500 bp band.

2. Shape of DNA: This is particularly true for plasmids that can be supercoiled, nicked, or linearized form. The supercoiled form moves the fastest, followed by linear and finally the nicked form which takes up the biggest space.

3. Concentration of Agarose: The higher the concentration, the smaller the pore size and vice-versa.

Source: https://www.minipcr.com/choosing-the-right-agarose-percentage/

4. Voltage Applied: The higher the voltage, the faster the movement and vice-versa. However, going too high will result in melting of the gel as well as irregular gel run pattern. The optimum voltage would be 5 V/cm.

5. Buffer Used: Some buffers have higher conductivity, hence helping for the faster movement of nucleic acids across the gel. Example, Tris-Borate EDTA (TBE) is better for resolving small fragments of DNA than larger fragments.

6. Type of Agarose: The mobility of agarose is also determined by the type of agarose used. For instance, low Electroendoosmosis (EEO) agar has better resolution and higher mobility compared to normal agarose.

7. Ethidium Bromide: Ethidium bromide (EtBr), used for staining the DNA for observation under the UV light (DNA bound by EtBr glows bright orange when exposed to UV light), increases the overall mass of the DNA due to its intercalating agent, hence, it can reduce the mobility of the nucleic acid when compared to those run in gel with the EtBr.