DNA Agarose Gel Electrophoresis

Agarose Gel Electrophoresis Electrophoresis is a molecular technique that separates nucleic acids and proteins based on: Size & charge In module one you watched a video to learn how to pour and load an agarose gel. Agarose gel electrophoresis is the most basic and common technique used in a biotechnology lab. Agarose is a gel matrix that separates DNA and RNA based on size, shape and charge.

Agarose Gel Electrophoresis DNA is a negatively charged molecule and therefore is attracted to positive charges. Where does the negative charge on DNA come from? Remember from module one that the backbone of DNA is made up of sugar and phosphate molecules that are held together by phosphodiester bonds. The phosphate molecules on the DNA backbone are negatively charged. These phosphate molecules confer a negative charge on the entire DNA molecule. Therefore, because opposites attract, when placed in an electrical field the negatively charged DNA will migrate toward the positive pole.

Agarose Gel Electrophoresis Agarose (complex carbohydrate) provides a matrix through which DNA molecules migrate. Larger molecules move through the matrix slower than small molecules The higher the concentration of agarose, the better the separation of smaller molecules Because all DNA is negatively charged, regardless of the length or source, the rate of DNA migration and separation through an agarose gel depends on the size of the DNA molecule. Agarose provides a gel matrix through which the DNA molecules migrate. Smaller molecules travel faster through the gel matrix than larger molecules. Think about it this way, if you pulled a series of nets across the center of a pool and released a tank full of snakes and worms in one end of the pool, the smaller worms will easily fit through the holes in your net and will get to the other end of the pool faster than the larger snakes which will get tangled in the net. It’s the same for DNA molecules. The smaller molecules are better able to navigate through the matrix and therefore travel faster than larger DNA molecules.

Agarose Gel Electrophoresis Electrophoresis Animation Click on the electrophoresis animation to visualize agarose gel electrophoresis.

Questions on Agarose Gel Electrophoresis What separates DNA fragments in gel electrophoresis? Why does DNA migrate towards the positive pole? Which moves faster? Small or large fragments? Why? How can determine the size of a DNA fragment using gel electrophoresis? What is the purpose of loading dye in gel electrophoresis? What is the purpose buffer in gel electrophoresis? What is the purpose of staining or using ethidium bromide in gel electrophoresis? What is the purpose of using different gel concentrations?