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Agarose Gel Electrophoresis: History, Principle, Requirements, Procedure, Applications


Agarose gel electrophoresis is one of the basic tools used in laboratories throughout the world for molecular biology research. If you need to confirm the presence of a PCR product, analyze restriction enzyme digestion, or validate plasmid integrity, agarose gel electrophoresis can do the job. Even with the advent of new methods such as capillary electrophoresis and next-generation sequencing, agarose gel electrophoresis is still irreplaceable because it is simple and inexpensive.

🧬 Table of Contents −

Figure 1: Workflow of Agarose Gel Electrophoresis(AI-generated illustration for educational purposes)

What is Agarose Gel Electrophoresis?

Agarose gel electrophoresis involves the separation of macromolecules, such as DNA, RNA, or proteins, on an agarose gel under electrical charge. Agarose, a type of polysaccharide isolated from the red algae agar, forms a porous gel structure when mixed with buffer and allowed to cool.

The movement of DNA fragments within the gel pores depends mainly on the fragment size. Small DNA fragments will move fast and travel far, while large DNA fragments will move slowly.

A Brief History:

Scientists developed gel electrophoresis concepts in the early 20th century. Arne Tiselius received the Nobel Prize in Chemistry in 1948 for his work on moving boundary electrophoresis. Researchers adapted the technique for DNA in the 1960s and 1970s, with agarose becoming the preferred medium for nucleic acids because of its large pore size compared to polyacrylamide (used for smaller fragments or proteins).

Principle of Agarose Gel Electrophoresis:

The nucleic acids, such as DNA and RNA, have a negative charge naturally because of the presence of the negative charges of the phosphates in the sugar-phosphate backbone of the molecule.

Since opposite charges attract each other, as a result, the negative charged DNA molecules move away from the negative terminal (black-colored) and travel towards the positive terminal (red-colored) under the influence of electric current.

The agarose gel provides a sieving matrix. The rate of migration depends on three main factors:

  1. Size: Smaller DNA fragments navigate the pores more easily and migrate faster.
  2. Charge: DNA has a roughly uniform charge-to-mass ratio, so separation occurs primarily by size.
  3. Conformation: Supercoiled plasmid DNA migrates faster than linear DNA of the same size, which migrates faster than relaxed circular forms.

The gel concentration determines pore size. Lower percentages create larger pores suitable for big fragments; higher percentages create smaller pores for better resolution of small fragments.

Materials Required:

  • Agarose powder
  • Electrophoresis buffer:
  1. TAE (Tris-Acetate-EDTA): Better for larger fragments (>2 kb) and when recovering DNA for downstream enzymatic reactions (e.g., ligation). Lower buffering capacity, so change buffer for long runs.
  2. TBE (Tris-Borate-EDTA): Superior resolution for smaller fragments (<2 kb), higher buffering capacity, sharper bands. Borate can inhibit some enzymes, so avoid if extracting DNA for cloning
  • DNA samples
  • DNA loading dye
  • DNA Marker(ladder)
  • Ethidium bromide or safe DNA stain
  • Gel casting tray and comb
  • Electrophoresis chamber and power supply
  • Micropipettes and tips
  • Measuring cylinder and conical flask
  • UV transilluminator

Procedure of Agarose Gel Electrophoresis:

Figure 2: Process visuals (AI-generated illustration for educational purposes)

Step-by-Step Procedure:

1.Preparation of 1000 mL (1 Litre) Electrophoresis Buffer:

1X TAE Buffer Preparation:

Composition of 1X TAE Buffer:

  • 40 mM Tris-acetate
  • 1 mM EDTA

Materials Required:

  • 50X TAE stock solution
  • Distilled water
  • Measuring cylinder or volumetric flask

Calculation:

Use the dilution formula:

C₁V₁ = C₂V₂

Where:

  • (C₁) = concentration of stock solution = 50X
  • (V₁) = volume of stock required
  • (C₂) = final concentration = 1X
  • (V₂) = final volume = 1000 mL

Calculation:

50 x V₁ = 1 x 1000

V₁ = 1000/50

V₁ = 20ml

Process:

  1. Take a clean 1 litre volumetric flask or bottle.
  2. Add 20 mL of 50X TAE stock solution.
  3. Add distilled water up to 1000 mL.
  4. Mix thoroughly.

The solution obtained is 1X TAE running buffer used for agarose gel electrophoresis.

Alternative: Preparation of 50X TAE Stock Solution (1 L):

Chemicals Required:

  • Tris base = 242 g
  • Glacial acetic acid = 57.1 mL
  • 0.5 M EDTA (pH 8.0) = 100 mL
  • Distilled water = up to 1000 mL

Process:

  1. Dissolve 242 g Tris base in about 700 mL distilled water.
  2. Add 57.1 mL glacial acetic acid.
  3. Add 100 mL of 0.5 M EDTA.
  4. Make the final volume up to 1000 mL with distilled water.
  5. Store at room temperature.

This stock solution is diluted to prepare 1X working buffer.

2. Preparation of Agarose Gel:

For a 1% Agarose Gel:

A 1% gel is commonly used for separating DNA fragments of 500 bp to 10 kb.

  • Agarose powder = 1 g
  • 1X buffer = 100 mL

Process:

a)       Add 1 g agarose into a conical flask containing 100 mL 1X TAE/TBE buffer.

b)      Heat/Boil the mixture  for 1–3 minutes until the agarose dissolves completely and the solution becomes clear.

c)       Allow the solution to cool to about 50–60°C.

d)     
Add DNA stain: Ethidium bromide = 0.5 µg/mL

Role of Ethidium Bromide (EtBr):

Ethidium bromide (EtBr) is used as a DNA staining agent in agarose gel electrophoresis. It binds to DNA by inserting itself between the nitrogen bases of DNA strands (intercalation). When exposed to ultraviolet (UV) light, EtBr fluoresces and makes the DNA bands visible. Ethidium bromide is carcinogenic/mutagenic and potentially harmful. Gloves and protective equipment should always be used while handling it.

Why do we boil/heat the Agarose Gel?

Boiling of the agarose solution is necessary because agarose does not dissolve in cold buffer solution at all; the mixture has to be boiled close to its boiling temperature (about 85°C to 95°C) for breaking the intermolecular bonds and dissolving the powder to get a clear solution. This solution will then slowly cool down to below 40°C temperature to allow the polymerized molecules to link themselves through hydrogen bonds, thereby making a homogenous 3D sieve. Dissolution of agarose ensures that there will be no clumps in the gel, leading to distorted results.

3. Casting the Gel:

a)       Seal the ends of the gel tray with tape or casting gates.

b)      Place the comb approximately 1–2 cm from one end of the tray.

c)       Pour the warm agarose solution slowly into the tray.

d)      Remove air bubbles using a pipette tip.

e)       Allow the gel to solidify for 20–30 minutes at room temperature.

4. Electrophoresis Chamber Setup:

a)       Remove the tape carefully.

b)      Place the gel tray into the electrophoresis tank.

c)       Add enough 1X running buffer to completely cover the gel.

5.Preparation of DNA Samples:

Mix:

·        5 µL DNA sample

·        1 µL loading dye

Loading dye contains glycerol, sucrose, or Ficoll, which makes the sample heavier so it settles properly into the well instead of floating in the buffer. And it also track DNA Movement. 

Commonly used: 6X Loading Dye:
A 6X loading dye is used because it is a concentrated form of loading dye that becomes the proper working concentration (1X) after mixing with the DNA sample.

6. Loading the Samples:

  1. Carefully place the micropipette tip into the well.
  2. Load:

·        DNA ladder = 5–10 µL

·        DNA samples = 5–20 µL

Avoid puncturing the bottom of the wells.

7. Running the Gel:

  1. Connect: Negative electrode (black) near the wells & Positive electrode (red) at the opposite end
  2. Apply electric current:- Usually 80–120 volts

       Running Time:

  • Approximately 30–60 minutes

DNA fragments migrate toward the positive electrode because DNA carries a negative charge.

Smaller DNA fragments move faster through the pores of the agarose gel, while larger fragments move more slowly.

Figure 3: Migration Mechanism (AI-generated illustration for educational purposes)

8. Visualization of DNA Bands:

a)       After electrophoresis, remove the gel carefully.

b)      Place it on a UV transilluminator or gel documentation system.

c)       Observe fluorescent DNA bands.

Observation:

  • Bright bands indicate DNA fragments.
  • Compare bands with the DNA ladder to estimate fragment size.
Figure 4: Result Visuals(AI-generated illustration for educational purposes)

Applications of Agarose Gel Electrophoresis:

  • Agarose gel electrophoresis is mainly used to separate DNA fragments based on their size. Smaller DNA fragments move faster through the gel pores than larger fragments.
  • It is used to check whether Polymerase Chain Reaction (PCR) amplification was successful by observing the amplified DNA bands.
  • Agarose gel electrophoresis helps in DNA fingerprinting for forensic investigations, paternity testing, and identification of individuals.
  • It is used to analyze DNA fragments produced after restriction enzyme digestion in molecular biology experiments.
  • Researchers use it to isolate and examine plasmid DNA during cloning and genetic engineering studies.
  • Agarose gel electrophoresis can help identify mutations or abnormal DNA fragments associated with certain genetic diseases.

Advantages of Agarose Gel Electrophoresis:

  1. Simple and easy to perform
  2. Cost-effective technique
  3. Efficient separation of DNA fragments
  4. Suitable for large DNA molecules
  5. DNA bands can be easily visualized

Disadvantages of Agarose Gel Electrophoresis:

  1. Lower resolution compared to PAGE
  2. Not suitable for very small DNA fragments
  3. Ethidium bromide is hazardous
  4. Agarose gels are fragile and can break easily
  5. UV light may damage DNA during visualization

Precautions:

  1. Use gloves while handling ethidium bromide.
  2. Avoid air bubbles during gel casting.
  3. Do not overload DNA samples.
  4. Ensure wells are properly formed.
  5. Always run DNA toward the positive electrode.
  6. Use fresh buffer for better results.
  7. Handle UV light carefully to avoid eye and skin damage.

References:

  1. Sambrook, J., & Russell, D. W. (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor Laboratory Press.
  2. Brown, T. A. (2016). Gene Cloning and DNA Analysis: An Introduction (7th ed.). Wiley-Blackwell.
  3. Green, M. R., & Sambrook, J. (2019). Agarose Gel Electrophoresis. Cold Spring Harbor Protocols.
  4. Lodish, H., Berk, A., Kaiser, C. A., et al. (2021). Molecular Cell Biology (9th ed.). W. H. Freeman and Company.
  5. Nelson, D. L., & Cox, M. M. (2021). Lehninger Principles of Biochemistry (8th ed.). W.H. Freeman.
  6. Bio-Rad Laboratories. “A Guide to Agarose Gel Electrophoresis.”
    Bio-Rad Laboratories
  7. Thermo Fisher Scientific. “DNA Gel Electrophoresis Protocol.”
    Thermo Fisher Scientific
  8. Agarose Gel Electrophoresis: Principle, Parts, Steps, UsesMay 14, 2024 by Sagar Aryal, PhD

 

 

 

 

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