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Agarose gel electrophoresis


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

Agarose electrophoresis is an important technique in genomic research. It separates biological molecules based on their size and charge. It is used for the separation of DNA, RNA, protein and even other biological molecules.

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 noble prize. 

Before going in-depth into the topic, we have to understand several terminologies such as what is electrophoresis? What are the different types of electrophoresis? How does electrophoresis work? And What are the applications of electrophoresis? let’s start with electrophoresis…

What is electrophoresis?

Any biological particles may be negatively charged, positively charged or neutral. We can characterize or separates particles based on their charge.

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

We can use electrophoresis for separation of any biological molecule based on their charge and we can identify that particular molecule.

Hence electrophoresis is a process used in the identification, separation and characterization of the 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 as 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 most reliable.

For example, if we want to separate DNA fragments with the difference of 5 to 10 bp, PAGE gives the best result as compared to horizontal gel electrophoresis.

As the agarose gel electrophoresis is commonly used for DNA separation. We will discuss agarose gel electrophoresis in detail.

Component of agarose gel electrophoresis

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

Agarose

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

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

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 increases the pore size is 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.

Agarose gel electrophoresis equipment

The agarose gel electrophoresis equipment contains the electrophoresis chamber, gel caster, gel comb, electrodes and clamps.

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 agarose gel electrophoresis equipment 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 DNA sample will be loaded. The positive and negative electrodes are attached on each side of the gel electrophoresis chamber.

Agarose gel electrophoresis buffer

Electrophoresis buffer serves as a liquid medium for migration of DNA into the gel. TAE and TBE are two different types of commonly used buffer in agarose gel electrophoresis. 


For more detail on how electrophoresis buffer helps DNA in migration, types of buffer and importance of electrophoresis buffer read our article: Agarose gel electrophoresis buffer


EtBr

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


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 


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 principle

The negatively charged DNA molecules migrate towards the positive charge under the influence of constant current, and 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

Agarose gel electrophoresis protocol

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. Once it reaches its boiling temperature the solution becomes clear which indicates that the agarose powder is dissolved into the buffer.

Carefully remove the flask from the oven and place if 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).

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.

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 into the buffer.

Take DNA samples and place them into the 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.

Connect the electrodes to the power pack and run the gel at 100v for 45 to 60min.

Agarose gel electrophoresis result

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.

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

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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. EtBr intercalates between DNA bases and emits fluorescent light. Orange coloured DNA bands are observed under the UV transillumination.

Application of Agarose gel electrophoresis

The agarose gel electrophoresis is widely employed in molecular genetics, more specifically 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 several disease diagnosis like 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.

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graphical representation of DNA and DNA ladder bands into the agarose gel.

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, covalently closed circular DNA and linear DNA can be easily identified by DNA gel electrophoresis.

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 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 the beautiful result.

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

 

Article covered by : Tushar Chauhan 

Article reviewed by: Binal Tailor

Categories: All articles, Explore, Gel electrophoresis Tags: , , , ,

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