Running an Agarose Gel - University of Leicester

Summary

This video explains the preparation and use of agarose gels in molecular biology for the separation and purification of nucleic acid fragments. It covers the process of mixing agarose powder with buffer, heating the mixture, and pouring it into a gel tray. The gel's concentration affects the separation of smaller or larger DNA fragments during electrophoresis. Important safety tips are provided when using ethidium bromide, a commonly used mutagenic stain. The procedure includes cooling the gel, adding a comb to form sample wells, and ensuring proper solidification before use.

Takeaways

• 🧪 Agarose gels are used in molecular biology to separate and purify nucleic acid fragments.
• 🔦 The fragments are made visible under ultraviolet light using staining techniques.
• 🧫 Samples are loaded into gel wells and separated by size through the application of an electric current.
• 📏 Agarose gels can separate fragments ranging from approximately 0.2 to 20 kilobases.
• ⚖️ The concentration of agarose in the gel (0.6%-3%) depends on the size of the fragments being separated; higher concentrations are better for smaller fragments.
• 🌡️ To make agarose gel, agarose powder and buffer solution are mixed and heated until the agarose dissolves completely.
• 🧤 Always handle the mixture with gloves during heating to avoid burns, and ensure the container is large enough to prevent overflow.
• 🔬 Ethidium bromide is commonly used to stain nucleic acids, but it is mutagenic and carcinogenic, requiring careful handling.
• 🧴 After the gel solidifies (around 20 minutes), remove the comb to create wells for loading the samples.
• ⏳ The gel solidifies when it turns from transparent to opaque; it must not be disturbed during this process to ensure uniform thickness.

Q & A

What is the primary use of agarose gels in molecular biology?

Agarose gels are primarily used in molecular biology for the separation and purification of nucleic acid fragments.

How can nucleic acid fragments in agarose gels be visualized?

Nucleic acid fragments can be visualized under ultraviolet light after staining.

How are the samples introduced into the agarose gel?

Samples are loaded into pockets, or wells, in the gel and separated by size using an electric current.

What is the typical size range of fragments that can be separated using agarose gel?

The size of fragments that can be separated using agarose gel typically ranges from 0.2 to 20 kilobases.

How does agarose concentration in the gel affect fragment separation?

Higher agarose concentrations help separate smaller fragments, while lower concentrations are better for separating larger fragments.

What are the main components of agarose gel?

The two main components of agarose gel are agarose, which is a white powder, and a buffer solution.

What is the process for preparing agarose gel?

Agarose is mixed with buffer solution and heated until the agarose fully dissolves. The solution is then cooled to around 60°C before pouring.

What precautions should be taken when using ethidium bromide as a stain?

When using ethidium bromide, gloves should always be worn as it is a mutagen and carcinogen. Care must be taken to avoid contamination.

How is the gel solidified after pouring?

The gel is poured into a tray with a comb to form wells, then left to solidify for about 20 minutes until it turns opaque.

What should be done if bubbles form during gel pouring?

If bubbles form, they should be moved aside with a disposable pipette tip, particularly near the comb, to avoid affecting the well shape.