Agarose Gel Electrophoresis: Definition, Principle, Process, Protocol and Applications

“Electrophoresis is a common genetic lab technique used to separate charged particles such as DNA or RNA based on their size.”


DNA has no color or smell and so we can’t see it. If we can’t find it, how can we analyze and process it? It’s hard, right! But what if somehow we paint our DNA or fragments of DNA, use its negative charge to run under an electrical field?

We can see DNA, not like the typical spiral/helical structure but we can observe it like a sharp band. That’s what agarose gel electrophoresis serves. It helps in seeing and running DNA, put simply. 

Painting and running can visualize the DNA and determine the size of DNA, respectively. And therefore it completes two of our objectives; checks presence of absence and finds the fragment size. 

 In the early 19th century, Johann Wilhelm Hittorf et al. stated that 

Under the influence of the electrical field, smaller organic ions can move through the aqueous solution and it is helpful in measuring properties and behavior of ions as well”.

This was the first evidence of electrophoresis reported.

After several years, Arne Tiselius, a Swedish biochemist first published a paper on the complete process and apertures of electrophoresis in 1937. He won the Nobel Prize in 1948.

Technically, what electrophoresis do is separates charged particles (like the DNA) on a gel and determines the size. 

The present technique has many applications in genetic studies. In fact, it’s the only technique that is needed in almost every genetic experiment. Every single experiment like,

  • gDNA isolation, 
  • PCR amplification, 
  • Mutation detection, 
  • Restriction digestion, 
  • Gene cloning
  • DNA sequencing, 
  • Gene amplification

Needs agarose gel electrophoresis to evaluate results. If you have a good hand in this, trust me you can do best in your research, projects, PhD and even in diagnostics. However, unlike the PCR, most steps in gel electrophoresis are manual. 

So it has several fantastic applications as well as limitations, that all we are going to discuss here. 

In the present blog post, I will explain each and every point associated with agarose gel electrophoresis such as definition, Principle, Process, Protocol, Applications and Optimizations. It will surely add more value to your knowledge of genetics. 

Stay tuned. 

What is electrophoresis?

The process of separating and characterizing molecules based on their size and charge is known as electrophoresis.

Definition:

Under the influence of the constant electrical current, the charged particle can move in a liquid or fluid medium, the entire process is known as electrophoresis.”

In short, electrophoresis is a process to identify, separate, and characterize biological molecules like DNA, RNA, or proteins. 

Aim and Objectives of gel electrophoresis: 

  • To identify biomolecules like DNA, RNA or proteins. 
  • To determine the size of biomolecules.
  • To run biomolecules under electrical current. 
  • To estimate the size of biomolecules 

Types of electrophoresis:

Usually, electrophoresis is of two types: vertical gel electrophoresis and horizontal gel electrophoresis.

Vertical gel electrophoresis runs samples upside down and discontinuously. Commonly, It is utilized in protein separation.  PAGE- polyacrylamide gel electrophoresis is a type of vertical gel electrophoresis that relies on Polyacrylamide instead of Agarose.

Horizontal gel electrophoresis runs samples continuously, parallel to the surface and separates DNA. It uses Agarose gel instead of Polyacrylamide. 

On the positive side, the horizontal gel electrophoresis is easier, reliable and provides the best results but it can’t separate fragments with smaller differences. Usually, to separate smaller difference fragments, PAGE is more reliable. 

For instance, to separate DNA fragments of length 5 to 10 bp, we need PAGE, not agarose gel. Contrary, to separate fragments of length 100 to 1000 bp we can use agarose gel. We will only discuss agarose gel electrophoresis for DNA and we will also not discuss the PAGE here. 

The present technique has another name which is Submarine Gel Electrophoresis as the entire gel remains covered with the running buffer, completely.

Utilities:

Chemicals: 

Agarose powder, TAE or TBE buffer, Ethidium bromide, and bromophenol blue dye.

Equipment:

Gel caster, electrophoresis chamber, voltage source, gel tray, comb, oven, Uv transilluminator.

Other utilities:

Pipettes, tips, flask, weight balance.

Time-duration:

70 minutes to 90 minutes.

The hypothetical representation of agarose gel electrophoresis equipment is shown below,

Graphical representation of the electrophoresis apparatus.
Graphical representation of the electrophoresis apparatus.

Principle of Agarose gel electrophoresis

As we said, the principle depends on the charge of particles. 

The negatively charged DNA molecules migrate towards the positive charge under the influence of constant current, thus the separation depends on the mass and charge of DNA. The DNA molecules are forced to move through the agarose gel pores. The rate of the migration depends on,

  • The strength of the field
  • The hydrophobicity of the DNA
  • The ionic strength of the buffer
  • The size and shape of the DNA
  • The temperature of the buffer

Three of the important factors that affect DNA migration rate are, 

  • Agarose concentration

If the concentration of agarose is lower, the DNA can migrate easily and faster and vice versa.

  • Confirmation of DNA

The supercoiled DNA migrates faster than the linear DNA and circular DNA.

The supercoiled form of DNA is more compact than other forms causing speedy migration.

  • Size of DNA

Larger DNA fragments run slower while smaller DNA fragments run faster. Shorter PCR amplicons migrate faster than the gDNA. 

Agarose gel electrophoresis Kit Components:

  • Agarose
  • Agarose gel electrophoresis instrument 
  • Agarose gel electrophoresis buffer
  • DNA Gel loading dye
  • EtBr
  • Powerpack
Equipment of agarose gel electrophoresis. Image credit: www.bio-rad.com

1. Agarose

Agarose is a polysaccharide extracted from seaweed.  A long chain (polymer) of agarobiose creates the agarose sugar. Removing Agaropectin from the Agarse forms Agarose. 

A linear agarose polysaccharide is made up of the monomeric unit of D-galactose and 3, a 6-anhydro-L-galactopyranose disaccharide.

The agarose power is water-soluble when boiling. When we boil it, it dissolves in the buffer and after cooling it forms hydrogen bonds (cross-linking) which results in polymerization. By forming hydrogen bonds between adjacent molecules, it creates a three-dimensional matrix of pores.

Higher the cross-linking; lower the pore size and vice versa.

The size of the pores varies as the concentration of agarose in the gel varies. The pores create a channel in the gel from where the DNA can migrate.

The melting temperature of the agarose is near the boiling temperature (~ 95ºC) whereas the gelling temperature is ~ 37ºC – 43ºC. As the concentration of agarose increases pores size decreases. For various DNA types, the concentration of agarose is given in the table below,

Concentration of Agarose

Types of DNA sample

approx. fragment size

0.8%

Genomic DNA

> 1 kb

1.0%

PCR product and plasmid DNA

400bp – 10kb

2.0%

PCR product

50bp – 2kb

3.0%

Restriction digestion

10bp to 1000bp

If the concentration of a gel and DNA fragment size doesn’t match, we can’t get good results. For instance, a larger DNA fragment can’t migrate in a highly concentrated gel. It produces a smear of DNA.

We can’t get a clear, distinguishable DNA band.   

The smaller molecules migrate faster than the larger molecules and therefore distinct DNA band patterns based on their size are observed.

2. Agarose gel electrophoresis equipment

The agarose gel electrophoresis equipment carries the electrophoresis chamber, gel caster, gel comb, electrodes, and clamps (labeled on the above figure).

The gel electrophoresis chamber is made up of high-quality acrylic. Agarose gel electrophoresis buffer is filled into the electrophoresis chamber.

The gel caster is an important component that is used to cast the gel. The clamp helps in placing the gel tray tighten into the gel caster so the gel can not leak out.

After the gel is settled down, the clamps are removed and the gel tray is transferred to the agarose chamber (which is filled with agarose gel electrophoresis buffer).

Gel combs make wells in agarose in which our DNA samples will be loaded. The gel electrophoresis chamber has a positive electrode on one end and a negative at another end.

The comb placing is shown in the figure below,

Comb setup and migration of DNA in a gel.
Comb setup and migration of DNA in a gel.

3. Agarose gel electrophoresis buffer

The buffer works as a liquid medium to migrate DNA well into the gel. TAE (Tris-acetate EDTA) and TBE (Tris-borate EDTA) are two common and widely used buffer systems. 

The buffer 

One of the main advantages of the electrophoresis buffer is its capacity to maintain the pH of the medium.

The borate present in the TBE buffer can react with the glycerol of the DNA gel loading dye therefore not the TBE but TAE buffer is usually the first choice to do electrophoresis.

Preparation of 10X TAE buffer (for 250ml)

  • 400mM Tris
  • 200mM acetic acid 
  • 10mM EDTA
  • Add D/W to make the final volume of 250ml

Note: use only a single type of buffer for both gel preparation as well as tank filling.

If you want to learn more about agarose gel electrophoresis buffers, please read our previous article. The article is dominating Google search and in the top 3 results of Google SERP. Read it here: Agarose gel electrophoresis buffer.

4. DNA gel loading dye:

DNA gel loading dye contains Bromophenol blue and glycerol. It monitors DNA migration in a gel while running. During the process, the dye runs ahead of the DNA so that we can measure the distance covered by DNA in a gel.

Also, the glycerol present in the DNA gel loading dye provides density to the DNA which makes DNA settle at the bottom of the well.

The composition of 1X loading dye,

  • 0.042 % (W/V) Bromophenol blue powder
  • 2.5 % Ficoll
  • 0.042% (W/V) Xylene cyanol FF (optional)
  • Final makeup with D/W

If you want to learn more about DNA gel loading dye, please read our previous article: DNA gel loading dye. This article is also giving a tough competition to other highly ranked articles.

5. EtBr:

Ethidium bromide is a fluorescent dye used for tagging DNA. DNA does not have its own color, EtBr intercalates with the DNA bases and gives fluorescent color under UV light. 

Caution: mutagenic alert!!

The EtBr is mutagenic. Wear gloves, a mouth cap and UV protective glass before handling.

We have covered an entire article about how EtBr intercalates with DNA, properties of EtBr, how to use EtBr and its importance. Read the article here: Role of EtBr in agarose gel electrophoresis.

6. Powerpack

Both electrodes are attached to the power pack. We can run DNA on 50V, 100V, or 120V depending upon the type of DNA sample. The powerpack is important in order to provide electric current during the process.  

Steps of gel electrophoresis:

The whole process can be divided into six steps:

  • Preparing gel
  • Gel casting
  • Sample loading
  • Running the gel
  • Visualizing the gel
  • Result interpretation or DNA purification.

Protocol & Process

  1. Preparing Agarose gel:

To separate PCR fragments 2% agarose gel is recommended. To prepare a 2% agarose gel, weigh 2.0 grams of agarose powder and put it in a flask.

Add 100ml of 1X TAE buffer (or TBE) in the flask and shake well until the agarose powder will mix into the buffer.

To prepare a 10X stock buffer and 1X working buffer, read our previous article: Agarose gel electrophoresis buffer.

Now heat the mixture into the oven at 95ºC temperature until the agarose gets dissolved completely in the buffer (or 3 to 5 minutes). Once it reaches its boiling temperature the solution becomes clear which indicates that the agarose powder is dissolved properly into the buffer.

Caution: Hot surface!!

Care must be taken while heating a gel in a microwave oven. If possible take necessary training before doing this.

Carefully remove the flask from the oven and put it for some time until it becomes touchable.

Now add 0.5μg/ml EtBr and shake well to mix it properly, (shake it gently so that it can’t form bubbles).

  1. Gel casting:

Place the gel tray on a gel caster and tighten it with the help of clamps. Put the gel comb on the gel tray.

Pour the gel into the gel caster and wait until it becomes solid.

Place it under some cool environment or you can also place it in the freeze to settle it faster.

Fill the electrophoresis chamber with the gel electrophoresis buffer and carefully place the gel tray into the chamber.

Now fill the remaining buffer, until it covers the whole gel. 

  1. Sample loading:

Take DNA samples, place them in chronological order and add BPB into it or mix it on the parafilm. For more detail on how to add DNA gel loading dye into DNA, read our previous article, the link is given somewhere in the article. 

Now carefully take 10μl of a sample and pour it into the well. Remember, do not break the gel while loading the sample.

Load every sample, orderly. Lastly, load the DNA ladder. You can also load the DNA marker or ladder in the first position. 

  1. Running the gel:

Connect the electrodes to the power pack and run the gel at 100v for 45 to 60min. Run the samples until the DNA covers a 75% distance towards the positive node. 

Red is a positive node and black is a negative node.

Remove the gel carefully, once the run is completed.

  1. Visualizing the gel:

Run the gel until the bromophenol blue (DNA gel loading dye) reaches up to the edge of the gel. Turn off the power supply, carefully remove the gel from the electrophoresis chamber, and drain off the buffer.

Note: The EtBr makes DNA visible.

Now place the gel carefully on the UV transilluminator platform and close the lead of the transilluminator.

Remember!! do not forget to wear gloves and eye-protective glasses, the Uv transilluminator emits UV light that can harm our skin cells.

 

The image represents DNA bands under UV transilluminator. Image credit: http://enfo.agt.bme.hu/drupal/en/node/8929

Turn on the UV light and the results should be like the image given above. EtBr intercalates between DNA bases and emits fluorescent light, we can observe orange DNA bands under UV transilluminator.

  1. Results interpretation:

A single DNA band is a makeup of similar types of fragments, the more the fragments, the more intense the band is. Our DNA ladder or maker helps to determine the fragment size.

See the figure below,

Agarose gel electrophoresis results
The graphical representation of DNA and DNA ladder bands into the agarose gel.

To understand results and interpret it, read this article: A complete guide for analyzing and interpreting gel electrophoresis results.

If downstream processing such as cloning, sequencing or anything else is involved, re-isolate DNA fragments from the gel. We will discuss DNA purification through the gel in some other articles.

The rate of the DNA migration is directly proportional to the size of it whereas the migration of DNA is inversely proportional to the concentration of agarose gel.  

Advantages of Agarose Gel Electrophoresis

The present technique is like a subsidiary genomic tool that helps researchers to validate results. The technique is, 

  • Accurate 
  • Reliable
  • Cost-effective
  • reproducible

Limitations of Agarose Gel Electrophoresis

The present technique has several shortcomings too. 

  • The use of current, carcinogens, chemicals, and heating steps, makes the present method unsafe and risky to use.
  • Moreover, we can’t determine the exact fragment size of DNA, it gives just a rough idea.
  • The process is time-consuming and lengthy.
  • It requires more manpower.
  • It can’t give us sequence information. 

Applications of Agarose Gel Electrophoresis:

As we said, the technique has many applications in genetics, especially in PCR, RFLP, AFLP and RAPD analysis. DNA fingerprinting technique also relies on agarose gel electrophoresis. Here we have listed and explained some of the fantastic applications, 

Identification and separation of gDNA: 

DNA extraction isolates gDNA/ genomic DNA, to validate the extraction (whether we get DNA or not) electrophoresis has been performed. Samples are run on 08% gel, well- distinguished bands show the quality of gDNA. 

It also indicates purity and allows us to purify the gDNA if required. gDNA contains the whole genomic DNA of a cell. Note that we don’t require a DNA ladder here. 

Validating PCR amplicons: 

How to know if your PCR has amplicons or not! Here electrophoresis helps to visualize amplicons, separates different fragments, determines fragments size and shows primer-dimer or non-specific amplification.  

Do use an appropriate size DNA ladder to estimate the size of DNA bands. 

DNA sequencing:

Yet another best application of gel electrophoresis is separating DNA fragments for DNA sequencing. We can’t sequence the whole genome for studying a single gene. 

To identify or sequence a gene or fragment, first, that fragment is amplified and separated on a gel. A fragment of our interest is isolated from a gel, purified and sent for sequencing. 

Restriction digestion: 

Restriction digestion greatly relies on electrophoresis. A gel identifies, separates and validates digested samples. We can get information if digestion occurred or not. To know more about restriction digestion click the link and read the article. 

In addition, it is used in restriction mapping and gene mapping too. 

Disease diagnosis: 

Diseases like Thalassemia, Sickle cell anemia, Cystic fibrosis or Hemophilia can directly be diagnosed by gel electrophoresis analysis. Well-separated fragments identify alleles, separates homozygous from heterozygous and finds mutation. 

Monogenic diseases can commonly be diagnosed easily. 

DNA topology studies: 

The present technique can even identify, separate and characterize different topological structures of DNA such as supercoiled, circular or naked DNA. 

DNA fingerprinting studies: 

DNA fingerprints allow us to separate individuals or organisms from biological ones. Markers like VNTR, STR or SSR are selected, amplified and separated to get the results. Running fingerprints on a gel makes it possible to identify individuals based on their DNA profile. 

To learn more, you can read this article: DNA fingerprinting

Besides, the agarose gel electrophoresis applies in other techniques, methods and fields are, 

  • DNA testing, 
  • Checking DNA quality 
  • Genome mapping, 
  • Distinguish homozygous and heterozygous 
  • Finds allele or allelic variations 
  • Marker assisted selection- RFLP, AFLP and RAPD
  • Phylogeny analysis

Optimization of Agarose Gel Electrophoresis:

I already mentioned the agarose gel electrophoresis protocol. It’s my standard protocol and you can use it for your experiments. 

  1. Use a TAE buffer whenever possible, The Borate in TBE can create a serious problem. 
  2. Run gel on appropriate current input, running at faster or slower speed create DNA smear or DNA diffusion in a gel, respectively. Do as per the protocol or advised by the manufacturer. 
  3. Use 0.8% gel for genomic DNA and 1.8% gel for PCR fragments. 
  4. Do not reuse the buffer. 
  5. Discard gel after use, do not reuse it. 
  6. Running DNA at higher voltage heats and gel and degrade DNA. 
  7. Do not use the gel if bubbles are seen. 
  8. Do not pour the sample if wells are broken. 

I personally prefer Seakem agarose powder which is cost-effective and gives a beautiful result.

Precautions: 

Some precautions are necessary for performing agarose gel electrophoresis:

  • Agarose powder is hazardous hence always wear gloves, face masks, and goggles while preparing an agarose gel.
  • During the boiling of agarose wear oven gloves (heat resistant). Do not use plastic gloves, it will burn your hand.
  • EtBr is carcinogenic and mutagenic therefore take necessary precautions before handling it.

Can we make the electrophoresis instrument in our lab?  think about it and let me know in the comment box. 

Summary:

  • Gel electrophoresis separates DNA on the basis of its charge and size. 
  • Increasing gel concentration decreases pore size and vice versa. 
  • Larger fragments run slower while smaller fragments run faster.
  • When the current turns on, the DNA starts moving to the positive node. 
  • The EtBr intercalates with DNA and emits fluorescence under UV light. 
  • BPB runs ahead of the DNA, monitors the migration. 
  • A DNA ladder helps to determine the size of DNA fragments.
  • The agarose gel electrophoresis often known as horizontal gel electrophoresis is used to separate nucleic acid (DNA/RNA) ranging between 50bp to ~15kb.  

Conclusion:

The agarose gel electrophoresis is a subsidiary technique that helps to determine DNA. The agarose gel electrophoresis is an unmatched and non-replaceable technique until now. However, some of the other fast and more accurate options are now available but are so costly.

Each step has a different type of risk for us and a different setup. It’s a time-consuming process, yet accurate. If you are a novice in this field, you should have to learn this technique for your successful research career. 

Though the process is complicated, once you learn it, you will enjoy doing it. 

FAQs 

What is the purpose of agarose gel electrophoresis? 

The purpose of doing agarose gel electrophoresis is to separate the charged particles like the DNA on the basis of their size. It determines the size of the biomolecule. 

What is the use of agarose in agarose gel electrophoresis?

The agarose powder used in the agarose gel electrophoresis process provides a matrix for DNA or RNA to pass through, under the influence of electric current. Pores of agarose polysaccharides allow DNA to go through. 

Why do we use Agarose gel electrophoresis?

To separate biomolecules like DNA, RNA or protein depending upon their size and charge, we use the gel electrophoresis technique. 

What is agarose made of?

Agarose is a type of polysaccharide made up of polymers of agarobiose.

What are the steps of Agarose gel electrophoresis? 

 A few steps of the technique are preparing the gel, Gel casting, sample loading, Running the gel, visualizing the gel and results & interpretation.

Sources:

Lee PY, Costumbrado J, Hsu CY, Kim YH. Agarose gel electrophoresis for the separation of DNA fragments. J Vis Exp. 2012;(62):3923. Published 2012 Apr 20. doi:10.3791/3923

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