Agarose Gel Electrophoresis - Animated Video

Summary

Agarose Gel electrophoresis is a method for separating DNA, RNA, and proteins by size and charge. It starts with preparing a gel with agarose powder and buffer, heated until dissolved, then cooled to 55°C. Ethidium bromide is added for DNA visualization under UV light. The gel is cast, solidified, and submerged in a running buffer. DNA samples with loading buffer are loaded into the gel wells alongside a DNA ladder for size reference. An electric current separates DNA fragments based on their size, with smaller fragments moving faster. The separated DNA is visualized as bands under UV light, allowing for size determination by comparison with the ladder.

Takeaways

• 🧪 Agarose Gel Electrophoresis is a technique used to separate DNA fragments based on size and charge.
• 🌡️ For DNA separation, low concentration gels are better for larger molecules, while high concentration gels are better for smaller ones.
• 🔬 Agarose must be heated to dissolve in a buffer like TAE or TBE, and then cooled to about 55 degrees Celsius before use.
• 🚫 Ethidium bromide, a mutagen and intercalating agent, is added to the agarose solution for DNA visualization under UV light and must be handled with care.
• 🌐 The electrophoresis tank is prepared with electrodes and a comb to create wells for sample loading.
• 🏺 A casting dam is used to contain the agarose solution during gel setting to prevent leakage.
• 🔋 The gel is submerged in a running buffer after solidification to provide ions for current and maintain pH.
• 🧬 DNA samples are prepared with a loading buffer that adds density and color, aiding in the loading process and monitoring separation.
• 📏 A DNA ladder, a molecular-weight size marker, is loaded alongside samples to provide a reference for determining DNA fragment sizes.
• ⚡️ An electric current is applied to pull DNA through the gel, with smaller fragments moving faster due to their charge and size.
• 🌌 DNA fragments are visualized as bands under UV light, allowing for the determination of their approximate sizes by comparison with the DNA ladder.

Q & A

What is agarose gel electrophoresis used for?

Agarose gel electrophoresis is used to separate DNA fragments or other macromolecules such as RNA and proteins based on their size and charge.

How does the concentration of agarose affect the separation of DNA fragments?

Larger DNA molecules are resolved better using a low concentration gel, while smaller molecules separate better at high concentration gels.

What is the role of a buffer in agarose gel electrophoresis?

An appropriate buffer like TAE or TBE is used to provide ions that carry a current and to maintain the pH at a relatively constant value during electrophoresis.

Why is the agarose solution heated?

Agarose does not dissolve until it is heated, so the solution is heated in a microwave oven to dissolve the agarose powder.

Why is ethidium bromide added to the agarose solution?

Ethidium bromide is an intercalating agent that binds to DNA and fluoresces under ultraviolet light, making the DNA bands visible during electrophoresis.

Why is it important to handle ethidium bromide with care?

Ethidium bromide is highly toxic and mutagenic, so it must be handled carefully, typically in a fume hood, to avoid exposure.

What is the purpose of the comb in the electrophoresis tank?

The comb is placed in the tank to create wells for loading samples onto the gel.

How does the loading buffer aid in the sample preparation for agarose gel electrophoresis?

The loading buffer, containing glycerol and dyes, adds density to the sample, allowing it to sink into the gel, provides color, simplifies the loading process, and allows the user to monitor the separation progress.

What is a DNA ladder and why is it used?

A DNA ladder is a molecular-weight size marker containing DNA fragments of known lengths, used as a standard reference to determine the sizes of unknown DNA fragments after electrophoresis.

How does the electric current affect the movement of DNA fragments in the gel?

An electric current is applied to pull the negatively charged DNA fragments through the gel towards the positive pole, with smaller fragments moving faster due to their size.

How can the approximate sizes of DNA fragments be determined after electrophoresis?

By comparing the positions of the DNA bands from the samples to the DNA ladder, their approximate sizes can be determined based on the known lengths of the ladder's fragments.