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Agarose Gel Electrophoresis Steps & Procedures

Annakay Newell, Greg Chin
  • Author
    Annakay Newell

    Annakay Newell has taught in the biological and environmental science fields for over ten years. She has a PhD in plant pathology from the University of Georgia, a MSc in plant pathology from the University of Arkansas and a BSc in Biology from the University of Arkansas Pine Bluff.

  • Instructor
    Greg Chin
Learn about gel electrophoresis. Understand what gel electrophoresis is, what it is used for, its steps, its procedure, how it works, and the apparatus used to perform the procedure. Updated: 12/15/2021

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Gel Electrophoresis

Gel electrophoresis is a laboratory technique that is used for the separation and analysis of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and proteins based on their molecular size or electric charge. Agarose gel electrophoresis is the most commonly used and effective method for the separation of DNA fragments. Electrophoresis equipment applies an electric charge to molecules that causes them to move towards the electrode that is oppositely charged. For example, DNA and RNA are negatively charged so they will migrate to the positively charged end of the gel.

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  • 0:08 Agarose Gel
  • 1:13 Gel Box and Power Supply
  • 2:42 Loading Buffer and Dye Front
  • 3:57 Ethidium Bromide and UV Box
  • 4:53 Lesson Summary
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What is Electrophoresis Used For?

Electrophoresis is used in a wide range of applications and in different sciences. Some of these include:

  • To analyze polymerase chain reaction (PCR) results- PCR is used to amplify DNA segments.
  • Gene analysis in the medical field- used to diagnose certain illnesses
  • Taxonomy - DNA profiling to differentiate species and evolutionary relationships
  • Paternity testing
  • Study of proteins
  • Macromolecule analysis using blotting techniques
  • DNA fingerprinting in criminal investigations

Gel Electrophoresis Apparatus

There are certain pieces of equipment that are necessary to complete the gel electrophoresis process. These include:

  • Agarose gel
  • Gel box
  • Power supply
  • Loading buffer
  • Stain
  • UV transilluminator

Agarose Gel

Gel electrophoresis setup begins with making an agarose gel. Agarose is a polysaccharide, containing galactose residues, that can be extracted from certain marine red algae (seaweed). It is a major component of the gel electrophoresis apparatus because it houses the molecules that are to be separated. Agarose is ideal for gel electrophoresis because it has a low gelling temperature, neutral charge, and forms stable gels. It is usually sold in powder form, and then can be dissolved in boiling water. Upon cooling, the mixture forms the agarose gel. Agarose can therefore be solid or liquid. Combs of desired sizes are usually used to embed wells into the agarose gel for loading of molecules that are to be separated. Molecules are usually separated in a straight line from where they were loaded, generally referred to as lanes. The agarose gel must be oriented in a specific way in a gel box to allow migration in the intended direction.

Agarose gel when solidified

Agarose gel seen in gloved hands

Gel Box and Power Supply

The power supply is a piece of equipment that connects to the gel box and provides controlled voltage and amperage to the system. The power supply is connected to the gel box by lead wires that are colored black (cathode/negatively charged electrode) and red (anode/positively charged electrode). These wires transfer the electric current from the power supply to the gel box. The black wire is attached to the rear of the gel box as DNA and RNA are negatively charged and will travel to the front of the gel box that has the positively charged red wire attached.

Gel box attached to a power supply by red and black lead wires

The gel box attached to a power supply by red and black lead wires seen here is part of the gel electrophoresis setup

Loading Buffer and Dye Front

Loading buffers are used for loading samples into the wells that are embedded in the agarose gel. Loading buffers are made of a colored dye and a density agent. The loading dye allows visibility and tracking of the sample as it migrates through the agarose gel. The density agent increases the density of the sample so that it sinks to the bottom of the well. As electrophoretic separation progresses, different dye lines are visible on the agarose gel. The dye front is the dye line that migrates the fastest, and it visually helps to ensure that the sample does not migrate off the end of the gel. It can be used to determine when the electrophoresis protocol is complete as the user can see how far down the gel the loading dye has migrated.

Agarose gel showing dye front

The dye front on agarose gel in this image is an important part of the gel electrophoresis process

Ethidium Bromide and UV Box

Ethidium bromide is an intercalating agent that is used to stain nucleic acids, acting as fluorescent tags for visualization after electrophoretic separation. It is the most commonly used dye for DNA and RNA. Because it binds with DNA, it is a highly toxic mutagen, and care is usually taken while handling it to prevent inhalation, ingestion, or skin absorption. Other dyes such as SYBER Green and GelRed have been used as alternatives to ethidium bromide. Agarose gels can be stained before or after electrophoretic separation. Stained molecules can be visualized using a UV box (ultraviolet transilluminator) because they are not visible to the naked eye. The ultraviolet radiation emitted from the UV box causes the dye to fluoresce. A UV box usually has a protective covering to protect the user from UV exposure. Facial screens are used in some cases.

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Frequently Asked Questions

What are the steps in gel electrophoresis?

The steps in gel electrophoresis includes:

  • Gel preparation
  • Sample and standard preparation
  • Running electrophoresis
  • Visualizing samples in the gel
  • Documenting results in the gel

What is the process of DNA gel electrophoresis used for?

Agarose gel electrophoresis is the most commonly used and effective method for the separation of DNA fragments. Electrophoresis equipment applies an electric charge to molecules that causes them to move towards the electrode that is oppositely charged. DNA is negatively charged so it will migrate to the positively charged end of the gel. DNA gel electrophoresis is used in many application such as fingerprinting, taxonomy, and PCR.

What is the principle of gel electrophoresis?

The term electrophoresis is a broad term that is used to describe the movement and separation of charged molecules when they are exposed to an electric current. Because the mobility of ions in an electric field varies, electrophoresis can manipulate these differences to separate them. The gel electrophoresis process includes using agarose gels to separate DNA, RNA, or proteins based on the principles of electrophoresis.

What are the 4 main components of gel electrophoresis?

The four main components of gel electrophoresis are the agarose gel, gel box and power supply, loading buffer and dye front, and ethidium bromide and UV box. These components work together to complete the electrophoresis process.

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