Gel Electrophoresis

 Key Points to Remember

• Gel electrophoresis separates DNA, RNA, and proteins based on size and charge.
• Molecules move through a gel under an electric field—smaller ones travel faster.
• Agarose gels are used for DNA/RNA; polyacrylamide gels (SDS-PAGE) for proteins.
• Ethidium bromide or Coomassie blue stains help visualize separated bands.
• It is widely used in genetic analysis, protein studies, and molecular biology research.

Keywords

Gel electrophoresis, agarose gel, SDS-PAGE, DNA separation, electrophoresis principle, RNA electrophoresis, molecular biology techniques, protein analysis.

Gel Electrophoresis

Definition

Electrophoresis is a laboratory technique used to separate and sometimes purify macromolecules such as proteins, DNA, and RNA based on their size, charge, and shape (conformation). It plays a vital role in molecular biology, biochemistry, and genetics research.

Purpose of Gel Electrophoresis

Gel electrophoresis is a method used to:

• Separate DNA fragments according to their size.
• Analyze or purify DNA, RNA, or proteins.
• Visualize and study nucleic acids or proteins in research and diagnostic laboratories.

In this process, samples such as DNA, RNA, or proteins are loaded into a gel matrix and moved under an electric field, allowing researchers to determine the molecular weight and purity of biomolecules.

Principle of Gel Electrophoresis

The technique works on the basic principle that charged molecules migrate in an electric field toward the electrode with the opposite charge.

• The gel acts as a molecular sieve containing small pores.
• Smaller molecules travel faster through these pores, while larger molecules move more slowly.
• DNA and RNA, being negatively charged due to their phosphate groups, move toward the positive electrode (anode).
• In the case of proteins, which may have different charges, a detergent called Sodium Dodecyl Sulfate (SDS) is used. SDS denatures the proteins, giving them a uniform negative charge so they migrate according to size during electrophoresis.

Types of Gel Electrophoresis

Feature	Agarose Gel Electrophoresis	Polyacrylamide Gel Electrophoresis (PAGE)
Gel Orientation	Poured horizontally	Poured vertically
Molecules Separated	Large molecules (e.g., DNA)	Small molecules (e.g., proteins, small DNA fragments)
Toxicity	Non-toxic	Neurotoxic (requires care)
Common Use	DNA and RNA separation	Protein and small DNA fragment separation
Staining	Before or after pouring	After polymerization
Common Stain	Ethidium Bromide	Coomassie Brilliant Blue

1. Gel Electrophoresis of DNA

Materials Required

• Ethidium Bromide (10 mg/ml) – for DNA visualization under UV light.
• Gel Loading Dye (6X) – contains tracking dyes and glycerol.
• 5X TBE Buffer (stock solution) – used as running buffer.
• Preparation (1 L):
• 850 ml distilled water
• 20 ml 0.5 M EDTA (pH 8.0)
• 54 g Tris base
• 27.5 g Boric acid
• Adjust to 1 L with water and autoclave.
• 0.5 M EDTA (pH 8.0)
• 46.5 g EDTA in 200 ml water; add NaOH pellets to dissolve and adjust pH to 8.0.

Sample Preparation

1.     Mix the DNA sample (plasmid, PCR product, etc.) with loading buffer.

2.     Use 1/5 volume of loading buffer per DNA sample.

3.     Mix well before loading onto the gel.

Steps to Pour and Run a DNA Gel

1.     Prepare 30 ml of 1% agarose gel using 1X TBE buffer.

2.     Heat in a microwave for about 15 seconds until dissolved.

3.     Cool slightly and pour into a casting tray with a comb to form wells.

4.     Allow the gel to solidify for 15 minutes.

5.     Place the gel into the electrophoresis chamber and add running buffer until submerged.

6.     Load the DNA samples carefully using a micropipette.

7.     Attach electrodes: DNA moves toward the positive (red) end.

8.     Run until the tracking dye approaches the bottom of the gel.

9.     Remove the gel and place it on a UV transilluminator to view DNA bands.

2. Gel Electrophoresis of RNA

Materials

• Gel Loading Buffer II – contains formamide, EDTA, SDS, xylene cyanol, and bromophenol blue.
• 10X Gel Loading Solution – 40% sucrose, tracking dyes.
• NorthernMax Formaldehyde Load Dye – for denaturing RNA.
• Ethidium Bromide (optional) – for visualization.

Procedure

1.     Prepare the Gel:

o    Dissolve 1 g agarose in 72 ml water, cool to 60°C.

o    Add 10 ml 10X buffer and 18 ml formaldehyde (37%).

o    Pour into the tray and insert a comb. Allow it to solidify.

2.     Prepare RNA Sample:

o    Add 0.5–3X volumes of Formaldehyde Load Dye to RNA sample.

o    Heat at 65–70°C for 5–15 minutes to denature RNA.

3.     Run the Gel:

o    Load samples and run at 5–6 V/cm until the dye migrates about two-thirds of the gel length.

o    Visualize RNA bands using a UV transilluminator.

3. SDS-PAGE for Protein Separation

Materials

• Stock Acrylamide Solution
• Running and Stacking Gel Buffers
• 10% Ammonium Persulfate (APS)
• 10% SDS
• Sample Buffer (Tris-HCl pH 6.8, SDS, sucrose, β-mercaptoethanol)
• TEMED (catalyst for polymerization)
• Protein Stain (Coomassie Blue) and Destaining Solution

Procedure

1.     Clean and assemble glass plates to form the gel cassette.

2.     Prepare the resolving (running) gel mixture and pour it between the plates.

o    Add a thin layer of butanol on top to prevent air exposure.

3.     After polymerization, discard butanol and pour the stacking gel. Insert a comb to create wells.

4.     Once set, mount the gel cassette in the electrophoresis tank.

5.     Fill with electrophoresis buffer and load protein samples.

6.     Connect to the power supply and run until the bromophenol blue dye reaches the bottom.

7.     Remove the gel, stain with Coomassie Brilliant Blue, and destain to visualize protein bands.

Applications of Gel Electrophoresis

• DNA fingerprinting and forensic analysis
• Checking PCR and cloning results
• RNA quality assessment before Northern blotting
• Protein purity and molecular weight analysis
• Diagnosis of genetic and infectious diseases

Safety and Handling Tips

• Always wear gloves and protective eyewear, especially when handling Ethidium Bromide or formaldehyde.
• Dispose of gels and buffers according to biosafety and chemical waste protocols.
• Avoid UV exposure during visualization—use a shield or camera system.

Conclusion

Gel electrophoresis is a powerful and versatile analytical technique used to separate and identify DNA, RNA, and proteins based on size and charge. Mastering this method is essential for students and researchers in molecular biology, biochemistry, and biotechnology.