Agarose gel electrophoresis: equipment, principle, protocol and application.

Electrophoresis is a common genetic lab technique used to separate charged particles such as DNA based on the size of the particle.

Because of this, the size of the DNA can be determined with the help of the electrophoresis.

Apart from their use in the separation of biomolecules, the electrophoresis technique such as PAGE can also be used in the DNA sequencing which determines the sequence of a DNA.

However, the process is now entirely automated.

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 behaviour 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. Later on in the year 1948, he was awarded the Noble Prize. 

Before going into the topic we have to understand the brief idea behind the gel electrophoresis and how it works.

The overall idea of DNA gel electrophoresis:

The brief explanation is as followed,

The DNA molecules separated on the basis of their charge and size.

As the concentration of agarose increases, the size of pore will decreases and vice verse. Therefore, the larger molecules cannot run faster in comparison with the smaller DNA fragments.

Because of this reason, The smaller fragments appear nearer to the positive charge (migrate faster towards positive node).

Once the electrical current passes through the gel, the DNA starts moving towards the opposite electrode.

A tagged fluorescent dye such as EtBr used to visualize DNA bands. Another dye named DNA gel loading dye monitors the migration of DNA by running ahead of it.

Furthermore, a known molecular weight DNA marker is used to calculate the exact size of DNA.

Once the DNA reached to the sufficient distance, the process is stopped to protect the DNA running out of the gel.

The agarose gel electrophoresis often known as horizontal gel electrophoresis is used to separate nucleic acid (DNA/RNA) ranging between 50bp to ~15kb.  

Key topics,

  • What is electrophoresis
  • Types of electrophoresis
  • Components of agarose gel electrophoresis
  • Principle of gel electrophoresis
  • The protocol of gel electrophoresis
  • Application of gel electrophoresis
  • Limitation of gel electrophoresis
  • Conclusion


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What is electrophoresis?

Any charged biological molecule can be characterized and separated using the electrophoresis method.

Under the influence of the constant electrical current, the charged particle can move in liquid or fluid medium, this process is called electrophoresis”.

Electrophoresis is a process used in the identification, separation and characterization of biological molecules.

Types of electrophoresis:

Majorly electrophoresis is of two types: vertical gel electrophoresis and horizontal gel electrophoresis.

Vertical gel electrophoresis runs sample from upside down and discontinuously. Generally, it is most suitable for protein separation and is called a PAGE (polyacryle amide gel electrophoresis).

Instead of agarose, the polyacrylamide is used in vertical gel electrophoresis.

Horizontal gel electrophoresis runs sample continuously, parallel to the surface and it is widely used for separation of DNA. Instead of polyacrylamide, agarose is used in horizontal gel electrophoresis.

Horizontal gel electrophoresis is easier, more reliable and gives best results. However, for the separation of the molecule with a smaller difference, PAGE is more reliable in comparison with agarose gel electrophoresis.

For example, if we want to separate DNA fragments with the difference of 5 to 10 bp, PAGE gives the best result, here the standard horizontal gel electrophoresis cannot be applicable. 

As the agarose gel electrophoresis is commonly used for DNA separation. We will discuss only the agarose gel electrophoresis in detail (not PAGE).

The agarose gel electrophoresis is also known as submarine gel electrophoresis because the entire gel remains covered with the running buffer, completely.

Agarose gel electrophoresis:

In the present section, we will discuss on the utilities, principle, time duration, procedure, preparation and protocol of agarose gel electrophoresis.

Utilities:

Chemicals: 

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

Equipments: 

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

Other utilities: 

Pipettes, tips, flask, weight balance.

Overall time: 

70 minutes to 90 minutes

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

Agarose gel electrophoresis equipment

Principle of agarose gel electrophoresis

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

As we discussed earlier if the concentration of agarose is lower, the DNA can migrate easily and faster and vice verse.

  • 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 and this is the reason for its speedy migration.

  • Size of DNA

If the size of a DNA is larger, the migration rate of it is lower and therefore PCR product of several hundred basepairs migrates faster than the total genomic DNA.

Component of agarose gel electrophoresis

  • Agarose
  • Agarose gel electrophoresis equipment 
  • Agarose gel electrophoresis buffer
  • DNA Gel loading dye
  • EtBr
  • Power pack

Equipment of agarose gel electrophoresis. Image credit: www.bio-rad.com

1. Agarose

Agarose is a polysaccharide extracted from the seaweed.  The long chain of (polymer) agarobiose creates the agarose sugar. Agaropectin is removed from the agar to form agarose.

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

The agarose powder is only soluble in the water on boiling. After cooling, it undergoes hydrogen bonding (cross-linking) which consequence in polymerization.  

By forming the hydrogen between an adjacent molecule, it creates a three-dimensional matrix of pores.

Higher the cross-linking lower the pore size and vice verse.

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 migrates.

The melting temperature of the agarose is nearby the boiling temperature (~ 95ºC) whereas the gelling temperature is ~ 37ºC – 43ºC.

As the concentration of agarose increased the pore size decreased. Depending upon the requirement of the type of DNA samples, the concentration of agarose is listed in the table:

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 gel is highly concentrated and DNA fragments are larger, the DNA fragment can not migrate into the gel and it creates a shearing type of band patterns.

The smaller molecules migrate faster than the larger molecules and therefore distinct patterns of DNA bands based on their size are produced.

2. Agarose gel electrophoresis equipment

The agarose gel electrophoresis equipment contains the electrophoresis chamber, gel caster, gel comb, electrodes and clamps (labelled 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 which 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).

The wells are formed with the help of gel comb in which the DNA sample will be loaded. The positive and negative electrodes are attached on each side of the gel electrophoresis chamber.

The comb placing is shown in the figure below,

Agarose gel electrophoresis

3. Agarose gel electrophoresis buffer

Electrophoresis buffer facilitates the liquid medium for the migration of DNA into the gel. TAE and TBE are two most commonly used buffer in the agarose gel electrophoresis

The electrophoresis buffer also maintains a constant pH during the run.

The TAE buffer is more preferable above TBE because the borate present in the TBE buffer reacts with the glycerol of the DNA gel loading dye.

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 on agarose gel electrophoresis, please read our previous article: Agarose gel electrophoresis buffer. The present article is also ranked high in the google search.

4. DNA gel loading dye:

DNA gel loading dye contains Bromophenol blue and glycerol.

The BPB runs ahead of the DNA. We can monitor the migration by gel loading dye.

Also, the glycerol present into the DNA gel loading dye gives density to the DNA which settled DNA 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 on 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 their own colour, EtBr intercalates with the DNA bases and gives fluorescent colour under UV light. 

Caution: mutagenic alert!! The EtBr is mutagenic. Wear gloves, mouth cap and UV protective glass before handing it.

We have covered an entire article about how EtBr intercalates with DNA, properties of EtBr, how to use EtBr and it’s important. Read the article here: Role of EtBr in molecular genetics and cytogenetics 

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. 

Agarose gel electrophoresis protocol

The entire procedure of agarose gel electrophoresis is divided into 6 parts:

  1. Preparation of gel
  2. Gel casting
  3. Sample loading
  4. Running the gel
  5. Visualizing the gel
  6. Result interpretation or DNA purification.

1. Preparation of Agarose Gel: 

For preparing 2.0% gel for PCR fragments, weigh 2.0 grams of agarose powder and put it in a flask.

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

For the preparation of 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 will dissolve into the buffer (or 3 to 5 minutes). Once it reaches its boiling temperature the solution becomes clear indicates that the agarose powder is dissolved into the buffer.

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

Carefully remove the flask from the oven and place 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 bubbles cannot be formed).

2. Gel casting:

Place the gel tray on to 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 the gel is covered by the buffer.

3. Sample loading:

Take DNA samples and place them into sequential 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 article: DNA gel loading dye

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

Load each sample in an order and, lastly, load the DNA ladder. You can also load the DNA marker or ladder at first position. 

4. 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 75% distance towards the positive node. 

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

After the completion of running, remove the gel carefully  

5. Visualizing the gel:

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

The EtBr is used to visualize the DNA.

Now place the gel carefully on the 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 result will be observed as shown in the figure above. EtBr intercalates between DNA bases and emits fluorescent light. Orange coloured DNA bands are observed under the UV transillumination.

6. Results interpretation:

A band is a compilation of a DNA fragment of similar type. The Know molecular marker,  known as DNA ladder is used 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.

We had written a beautiful article on interpretation and analysing gel electrophoresis results. You can read this article here: A complete guide for analysing and interpreting gel electrophoresis results

If your experiment involved some of the downstream application such as cloning, we need to purify the DNA. For that, the DNA fragment is re-isolated or extracted from the gel.

We will discuss DNA purification through the gel in some other article.

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.  

Application of Agarose gel electrophoresis

The agarose gel electrophoresis is widely employed in molecular genetics, especially in PCR and PCR based techniques such as DNA fingerprinting, RFLP, AFLP and RAPD analysis, analysis of genetic markers, VNTR analysis.

Agarose gel electrophoresis is commonly used in the diagnosis of several diseases such as thalassemia, sickle cell anaemia, haemophilia, cystic fibrosis and other mutation analysis.

Further, it is used in restriction digestion based studies such as restriction mapping, genomic mapping and restriction digestion of cDNA etc.

Additionally, it allows purification of DNA fragments and separation of DNA fragments for sequencing and other downstream application.

Agarose gel electrophoresis is applicable to the study of DNA topology. Supercoiled, circular, nicked and linear DNA can be identified using the agarose gel electrophoresis. 

Limitations of Agarose gel electrophoresis

The present interpretation method is so useful but it has several limitations.

The risk factor is very high in gel electrophoresis due to the use of current, UV light, carcinogenic chemical and heating procedures.

Also, the DNA fragment size determined is not accurate (it is just an assumption).

The process is time-consuming and lengthy.

Furthermore, sequence information cannot be obtained by using it.

My ultimate guide using agarose gel electrophoresis

I already mentioned the agarose gel electrophoresis protocol. You can use agarose gel electrophoresis protocol in your routine lab and it is our standardized protocol.

Some precautions are necessary for performing agarose gel electrophoresis:

  • Agarose powder is hazardous hence always wear gloves, face mask and goggles while preparing of 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 a carcinogenic and mutagenic therefore take necessary precautions.

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

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


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Conclusion:

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.

The entire process of electrophoresis simple but each step carries a different type of risk, therefore, care must be taken every time while preparing or doing agarose gel electrophoresis.