Running an Agarose Gel - University of Leicester

University of Leicester

26 Jun 200907:13

Summary

TLDRThis 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.

Outlines

00:00

🔬 Agarose Gels in Molecular Biology

Agarose gels are crucial tools in molecular biology, primarily used for the separation and purification of nucleic acid fragments. These fragments become visible under ultraviolet light after staining. Samples are loaded into gel wells and separated by size using electric current. Agarose gels can separate fragments ranging from 0.2 to 20 kilobases in size. The concentration of agarose, which varies between 0.6-3%, affects the separation process—higher concentrations are used for smaller fragments, while lower concentrations are ideal for larger ones. The gel preparation requires two main components: agarose, a white powder, and a buffer solution. The mixture is heated to create the gel.

05:01

🧪 Preparation of Agarose Gel

To make agarose gel, the correct amount of agarose and buffer solution is measured based on the desired gel size and concentration. The agarose is mixed with the buffer solution, ensuring the mixture is lump-free, and then heated in a microwave until the agarose fully dissolves. During heating, it's important to use a container large enough to prevent the solution from boiling over, with a loosely placed lid to avoid pressure buildup. The solution should be stirred periodically while wearing protective gloves, as overheating can cause sudden boiling. Full dissolution is indicated by the solution becoming completely transparent. Once dissolved, the mixture should cool to around 60°C to avoid damaging the gel container while keeping the gel in liquid form.

🎨 Adding Dye to Agarose Gel

Once cooled, a dye is added to the molten gel to visualize nucleic acid fragments after separation. Common dyes include Sybr Green, though the most frequently used is ethidium bromide. When using ethidium bromide, precautions are necessary as it is a mutagen and carcinogen, capable of entering the body through skin or inhalation. It is vital to change gloves immediately after handling ethidium bromide to prevent contamination of other lab equipment.

🛠️ Pouring the Agarose Gel

To pour the gel, the ends of the gel tray need to be sealed, typically using masking tape, to prevent leakage. The tape should extend beyond the tray's edges and be pressed down firmly. The tray must be placed on a level surface to ensure an even gel thickness. A comb is inserted into the tray to create wells, or pockets, where nucleic acid samples will be loaded. Once the tray is prepared, the liquid gel is slowly poured in, and any air bubbles, especially around the comb, should be removed using a pipette tip.

⏳ Setting and Removing the Gel

Ensuring no bubbles remain near the comb is crucial for creating properly shaped wells. The gel solidifies over about 20 minutes, becoming opaque in the process. During this time, it is important to avoid moving the gel tray or touching the gel, as this could result in uneven thickness. Once the gel has set, the comb is carefully lifted straight up to avoid damaging the wells, and the masking tape is removed from the ends of the tray.

Mindmap

Keywords

💡Agarose Gel

Agarose gel is a substance used in molecular biology for separating and purifying nucleic acid fragments. It is prepared by mixing agarose powder, a polysaccharide, with a buffer solution, and heating it until fully dissolved. In the video, agarose gel is central to the process, as it acts as the medium through which nucleic acid fragments are separated based on size using an electric current.

💡Nucleic Acid Fragments

Nucleic acid fragments refer to segments of DNA or RNA that can be separated by size using agarose gel electrophoresis. In the video, these fragments range in size from 0.2 to 20 kilobases and are made visible after separation using staining agents under ultraviolet light. The separation of these fragments is a crucial step in many molecular biology techniques.

💡Electrophoresis

Electrophoresis is the technique used to separate nucleic acid fragments based on size by applying an electric current. The nucleic acids move through the agarose gel matrix toward the positive electrode, with smaller fragments moving faster than larger ones. This process is a key focus of the video, demonstrating how the gel separates DNA or RNA fragments effectively.

💡Agarose Concentration

Agarose concentration refers to the amount of agarose used in the gel, which affects the resolution of nucleic acid fragment separation. In the video, different concentrations (0.6-3%) are mentioned, with higher concentrations being more suitable for smaller fragments, and lower concentrations for larger ones. This is crucial for optimizing the gel for different experimental needs.

💡Buffer Solution

Buffer solution is the liquid component mixed with agarose to form the gel, and it maintains a stable pH during electrophoresis. The video explains how the buffer ensures the gel functions properly, and its ionic properties are essential for conducting the electric current needed for fragment separation.

💡EtBr (Ethidium Bromide)

Ethidium bromide (EtBr) is a commonly used fluorescent dye in molecular biology, mentioned in the video as a mutagenic and carcinogenic substance. It binds to nucleic acids and, when exposed to ultraviolet light, makes the separated DNA fragments visible. The video emphasizes the safety precautions necessary when handling EtBr, including the need for gloves and careful disposal.

💡Microwave Heating

Microwave heating is a method used to dissolve agarose in the buffer solution during gel preparation. The video describes how the mixture should be heated until fully transparent, with intermittent stirring to prevent overheating. The process is crucial for ensuring the agarose fully dissolves, enabling an even and functional gel for electrophoresis.

💡Gel Casting

Gel casting refers to the process of pouring the agarose solution into a mold, or gel tray, to form the gel used in electrophoresis. The video outlines steps like sealing the tray’s open ends with tape and inserting a comb to create wells for loading samples. This part of the video focuses on the practical aspects of preparing the gel for the experiment.

💡Comb and Wells

The comb is a tool placed into the agarose gel before it solidifies, creating wells (small indentations) where the nucleic acid samples are loaded. The video explains that these wells are formed at the top of the gel and are essential for holding the DNA or RNA samples during electrophoresis. Ensuring the comb is inserted properly helps maintain the integrity of the wells.

💡UV Light Detection

UV light detection is a method used to visualize nucleic acid fragments after electrophoresis. In the video, it explains how after the separation process, the stained DNA fragments can be illuminated and photographed using ultraviolet light. This step is critical for analyzing the results of the experiment and determining the size of the nucleic acid fragments.

Highlights

Agarose gels are used in molecular biology to separate and purify nucleic acid fragments.

Fragments can be visualized under UV light after staining.

Samples are loaded into the gel pockets and separated by size using electric current.

Agarose gels can separate fragments ranging from 0.2 to 20 kilobases.

Gel concentration varies between 0.6% and 3%, depending on the size of the fragments to be separated.

Higher agarose concentrations improve separation of smaller fragments, while lower concentrations are better for larger fragments.

Agarose gels consist of two main components: agarose powder and buffer solution.

To prepare the gel, agarose is dissolved in the buffer by heating, ensuring it is fully dissolved and clear.

Incomplete dissolution of agarose can lead to uneven concentration in the gel, affecting the separation of nucleic acid samples.

The agarose solution should be cooled to 60°C before pouring to avoid damaging the gel tray.

Stains like Sybr Green or ethidium bromide are added to the gel to visualize the separated nucleic acid fragments.

Ethidium bromide is a mutagen and carcinogen, requiring gloves during use, and contaminated gloves must be discarded immediately.

Masking tape is used to seal the open ends of the gel tray before pouring the gel, which ensures an even thickness.

A comb is inserted into the gel tray to form pockets, which will later hold the nucleic acid samples.

Once the gel solidifies, the comb is carefully removed, and the gel is ready for use.