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.

Outlines

00:00

🧬 Agarose Gel Electrophoresis: DNA Fragment Separation

Agarose Gel electrophoresis is a method for separating DNA fragments based on size and charge. The process begins with preparing a gel by weighing agarose powder and adding a buffer like TAE or TBE. The mixture is heated to dissolve the agarose and then cooled to 55 degrees Celsius. While cooling, an electrophoresis tank with electrodes is set up, and a comb is placed to create wells. Ethidium bromide, a mutagen and intercalating agent, is added to the solution before pouring it into the tank to facilitate visualization under UV light. The gel solidifies, and the comb is removed. DNA samples, prepared with a loading buffer for density and color, are loaded into the wells alongside a DNA ladder for size reference. An electric current is applied, causing negatively charged DNA fragments to move towards the positive pole, with smaller fragments moving faster. The process is monitored and stopped before the dye exits the gel. Finally, the gel is examined under UV light to visualize the separated DNA bands.

05:03

🔍 DNA Fragment Size Determination

After the electrophoresis process, the DNA fragments are separated into distinct bands on the agarose gel. By comparing the position of these bands to the DNA ladder, which contains fragments of known lengths, the approximate sizes of the unknown DNA samples can be determined. This comparison allows for the assessment of the molecular weight of the DNA fragments, providing valuable information for further analysis and research.

Keywords

💡Agarose Gel Electrophoresis

Agarose Gel Electrophoresis is a technique used to separate DNA fragments or other macromolecules based on their size and charge. It is central to the video's theme as it is the primary method discussed for DNA fragment separation. In the script, it is described as the process starting with gel preparation and leading to the visualization of DNA bands under UV light.

💡Gel Preparation

Gel preparation, also known as gel casting, is the first step in agarose gel electrophoresis. It involves weighing out agarose powder and dissolving it in a buffer to create a gel matrix. This step is crucial as the concentration of the gel affects the resolution of DNA fragments, with low concentrations for larger molecules and high concentrations for smaller ones.

💡Buffer

A buffer, such as TAE or TBE, is a solution used in electrophoresis to maintain a stable pH and provide ions necessary for conducting electricity. The script mentions pouring an appropriate buffer into the bottle with agarose powder, highlighting its importance in the electrophoresis process.

💡Ethidium Bromide

Ethidium Bromide is a highly toxic mutagen used as an intercalating agent in agarose gel electrophoresis. It binds between the base pairs of DNA, and when exposed to UV light, it fluoresces, making DNA bands visible. The script warns of its toxicity and the need for careful handling, emphasizing safety in laboratory practices.

💡DNA Ladder

A DNA ladder is a molecular-weight size marker used as a reference in electrophoresis. It contains DNA fragments of known lengths and is loaded into the gel to help estimate the size of unknown DNA fragments. The script describes it as a standard reference, illustrating its role in comparing and sizing DNA bands.

💡Loading Buffer

Loading buffer is added to DNA samples before electrophoresis. It contains glycerol and dyes like bromophenol blue, which add density to the sample, facilitate its loading into the gel, and allow for monitoring the progress of separation. The script mentions its use before sample loading, showing its utility in the electrophoresis procedure.

💡Molecular Weight

Molecular weight refers to the mass of molecules, such as DNA fragments, and is a key factor in their migration during electrophoresis. Smaller molecules, having less mass, move faster through the gel than larger ones. The script discusses how DNA molecules, based on their charge and size, travel at different speeds in the gel.

💡Negative Electrode

The negative electrode is one of the two electrodes used in the electrophoresis tank. It is part of the electric field that drives the negatively charged DNA fragments towards the positive electrode. The script mentions preparing the electrophoresis tank with electrodes, indicating the setup's role in the separation process.

💡DNA Fragments

DNA fragments are pieces of DNA that are separated during agarose gel electrophoresis based on their size and charge. The script describes the process of these fragments moving through the gel towards the positive pole, illustrating the principle of size-based separation in the technique.

💡UV Light

UV light is used to visualize DNA bands in agarose gel electrophoresis after the separation process. The script explains that once the DNA fragments have been separated, the gel is placed under UV light to make the bands visible, highlighting the final step in analyzing DNA samples.

💡Sugar-Phosphate Backbone

The sugar-phosphate backbone is a structural component of DNA, composed of alternating sugar (deoxyribose) and phosphate groups. It gives DNA its negative charge, as mentioned in the script, which is essential for the migration of DNA fragments in an electric field during electrophoresis.

Highlights

Agarose Gel electrophoresis is used to separate DNA fragments based on size and charge.

Larger DNA molecules are resolved better using a low concentration gel.

Smaller DNA molecules separate better at high concentration gel.

Agarose powder is weighed and mixed with an appropriate buffer like TAE or TBE.

The agarose solution is heated until fully dissolved.

The solution is cooled to about 55 degrees Celsius before pouring.

An electrophoresis tank with electrodes is prepared for gel casting.

Ethidium bromide is added to the agarose solution for DNA visualization under UV light.

Ethidium bromide is highly toxic and must be handled with care in a fume hood.

DNA samples are prepared with a loading buffer for density and color.

A DNA ladder of known lengths is used as a molecular-weight size marker.

DNA fragments move towards the positive pole due to their negative charge.

Smaller DNA fragments move faster through the gel than larger ones.

Bromophenol blue migrates faster than DNA and is used to stop electrophoresis before complete migration.

DNA fragments are visualized as bands under UV light after separation.

Each band represents a group of DNA fragments of the same size.

The DNA ladder is used to determine the approximate sizes of the DNA bands.

Transcripts

Agarose Gel electrophoresis is a technique

used to separate DNA fragments or other macromolecules

such as RNA and proteins based on their size and charge

The first step for DNA fragments separation

is the gel preparation or gel Casting

An appropriate amount of agarose powder is weighed out

For standard agarose gel electrophoresis

larger molecules are resolved better using a low concentration gel

while smaller molecules separate better at high concentration gel

Once the agarose has been weighed

an appropriate buffer such as TAE or TBE is poured into the bottle

agarose does not dissolve until it is heated

Therefore, the solution is heated for few seconds in a microwave oven

Once the agarose is fully dissolved

the solution is cooled to about 55 degrees Celsius in a water bath

While the agarose solution is cooling

an electrophoresis tank, with a negative electrode and a positive electrode is prepared for gel casting

A comb is placed in the tank to create wells for loading samples

then casting dams are fixed in the tank

so that the agarose solution does not flow out during setting

once the agarose solution has cooled to about 55 degrees celsius

and before pouring it, into the electrophoresis tank, ethidium bromide is added

ethidium bromide is highly toxic as a mutagen

so it must be carefully handled in a fume hood

Ethidium bromide is an intercalating agent

which intercalates between base pairs of the DNA molecule

When it is exposed to ultraviolet light, it will fluoresce

and DNA bands become visible

The more DNA present, the brighter the band

after addition of ethidium bromide

the agarose solution is poured into the electrophoresis tank

then, the gel is allowed to solidify at room temperature

Once the gel has solidified, the comb is carefully removed

as well as the casting dams

Next, the gel is submerged in a TAE or TBE running buffer

which used to provide ions that carry a current

and to maintain the pH at a relatively constant value

After the preparation of the agarose gel

the next step is the preparation of the DNA samples

A loading buffer is added to each sample

This buffer contains a glycerol and some dyes such as bromophenol bleu

And it is used to add density to the sample, allowing it to sink into the gel

to provide color and simplify the loading process

and to allow the user to monitor the progress of the separation

before loading the samples into the wells

a molecular-weight size marker known as a DNA ladder

is commonly used as a standard reference that contains DNA fragments of known lengths

it is loaded into the first well of the agarose gel

Once the molecular-weight size marker has been loaded

the DNA samples are loaded into the wells

After adding the samples into the wells

a lid is placed on the electrophoresis tank

Then, an electric current is applied to pull the samples through the gel

Based on their charge and size

the DNA molecules will travel through the gel at different speeds

The DNA molecules have a negative charge

because of the phosphate groups in their sugar-phosphate backbone

herefore, when placed in an electric field

DNA fragments start moving through the matrix of the gel towards the positive pole

Because all DNA fragments have the same amount of charge per mass

small fragments move through the gel faster than large ones

Due to the relatively small molecule size of bromophenol blue, it migrates faster than DNA

and by optical control of the migrating-colored band, the electrophoresis can be stopped before the dye

so before the samples have completely migrated through the gel and leave it

Once the DNA fragments have been separated

the gel is placed under UV light

Then, the DNA fragments can be seen as bands.

Each band contains a large number of DNA fragments of the same size

that have all traveled as a group to the same position

By comparing the DNA bands of the samples to the DNA ladder

we can determine their approximate sizes