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Ethidium Bromide, Loading Buffer & DNA Ladder: Visualizing DNA and Determining its Size Video

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  • 0:09 Gel Electrophoresis Review
  • 1:10 Ethidium Bromide
  • 2:43 Loading Buffer
  • 4:53 DNA Ladder
  • 6:54 Lesson Summary
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Lesson Transcript
Instructor: Greg Chin
In this lesson, you will learn about the role that ethidium bromide, loading buffers, and DNA ladders play in visualizing DNA and determining the size of DNA fragments in agarose gel electrophoresis.

Gel Electrophoresis Review

Agarose gel electrophoresis plays a critical role in analyzing DNA in laboratory experiments. It is a method of separating biological molecules using an electrical current. We learned previously that DNA molecules can worm their way through the pores in an agarose gel. The negative charge of DNA molecules propels them through the electrical field toward the positive electrode. Shorter DNA molecules can travel farther in an agarose gel in a given amount of time than longer counterparts. Agarose gel electrophoresis provides a means of analyzing DNA by separating molecules based on size. However, in and of itself, agarose gel electrophoresis does not provide a means of visualizing the DNA. That role is played by two dyes: ethidium bromide and loading buffer.

Ethidium Bromide

Ethidium bromide binds to DNA and fluoresces under an ultraviolet light
Ethidium bromide UV light

Ethidium bromide is a molecule commonly used to visualize DNA in agarose gel electrophoresis experiments. It both binds to DNA and fluoresces under the proper conditions. Ethidium bromide is known as an intercalating agent. The flat structure of ethidium bromide allows it to intercalate, or insert, between nitrogenous bases of a DNA molecule. This interaction is especially useful because of ethidium bromide's second characteristic. When it is exposed to ultraviolet light, ethidium bromide fluoresces. Thus, this chemical provides both a means of tagging DNA molecules and a means of visualizing them.

However, note that any molecule that binds to or in any way alters the molecular structure of DNA can be dangerous to a living organism. Although there is some debate about the exact nature of the health risk posed by exposure to ethidium bromide, most organizations and agencies do consider it a risk. Because of the intercalating characteristic of ethidium bromide, it is believed by many to pose a mutagenic risk. However, with strong safety protocols in place, ethidium bromide continues to be one of the most common methods of visualizing nucleic acids in agarose gel electrophoresis experiments.

Loading Buffer

Well, that's the end of the story, right? Agarose is going to separate DNA molecules based on size, and ethidium bromide is going to tag the molecules so we can visualize them under UV light. Well, not exactly. DNA, like many biological molecules, is colorless. To complete the electrical circuit, the gel must be bathed in a salt solution called electrophoresis buffer. Water is also colorless. See the problem?

Loading buffer is a solution added to a DNA sample to make it visible to the naked eye
loading buffer

If DNA is colorless and the electrophoresis buffer bathing the agarose gel is also colorless, how are we going to tell if we've inserted the DNA sample into the well of the gel or not? We could add ethidium bromide and perform the entire protocol under UV light, but that seems unnecessarily dangerous to our health. The answer is that we need to use a different solution, called loading buffer. Loading buffer is added to a DNA sample to give it color to the naked eye.

Notice that monitoring the progression of the dye molecules in the gel will also allow us to determine when the protocol is complete. Fantastic. Now I can tell if I've accurately inserted our DNA sample into the well. But there's still one more problem. DNA is an aqueous molecule.

That means that it is hydrophilic and will dissolve in water. If our DNA samples dissolve in the water of the electrophoresis buffer, the DNA molecules will be spread evenly throughout the solution and will not stay localized to the given well.

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