Agarose gel electrophoresis is a technique used in biochemistry, molecular biology, genetics, and clinical chemistry.
It separates DNA, RNA, or proteins based on size and charge using a gel matrix made of agarose.
Agarose is a natural linear polymer extracted from seaweed; it forms a gel through hydrogen bonding when heated in buffer and cooled.
Agarose gels are the preferred medium for separating moderate to large-sized nucleic acids.
This method is widely used due to its broad separation range and ease of use.
Gel electrophoresis is a fundamental technique in molecular biology laboratories.
It is primarily used for nucleic acid (DNA/RNA) and protein analysis.
Key applications include:
Checking PCR results
Estimating the size of DNA fragments
Analyzing genetic variations
Preparing DNA for downstream experiments
The principle is based on the movement of charged molecules through the gel under an electric field.
Agarose gel electrophoresis is most commonly used for DNA analysis.
Principle of Agarose Gel Electrophoresis
Gel electrophoresis separates DNA fragments based on size using a gel matrix (typically agarose).
DNA samples are pipetted into small wells in the gel.
An electric current is applied, causing negatively charged DNA (due to phosphate backbone) to migrate toward the positive anode (+).
The rate of migration is inversely proportional to fragment size:
Smaller DNA fragments migrate faster and travel farther.
Larger DNA fragments migrate slower and remain closer to the wells.
The agarose gel functions as a molecular sieve, allowing separation based on size.
DNA is stained with an intercalating dye such as ethidium bromide (or alternatives like SYBR Safe).
As DNA moves, it incorporates the dye, and upon UV illumination, the stained DNA fluoresces.
Fluorescence intensity depends on DNA fragment size:
Larger fragments fluoresce more intensely due to more dye uptake.
Smaller fragments show less intense fluorescence.
A DNA ladder (molecular weight marker) with fragments of known sizes is run alongside the samples:
It is used to estimate the sizes of unknown DNA fragments.
Instrumentation & Equipment Needed
Electrophoresis chamber – holds the gel and buffer, connected to the power supply.
Power supply unit – applies the electric field (adjustable voltage and timer).
Gel casting tray – UV-transparent tray used to cast the agarose gel.
Sample combs – create wells in the gel for sample loading.
Gel electrophoresis tank – holds the gel and buffer during the run.
Transilluminator (UV light box) – visualizes stained DNA bands post-electrophoresis.
Heating source – such as a microwave or heating plate, to melt agarose.
Chemicals & Reagents
Agarose powder – ultrapure grade for clear gel formation.
Electrophoresis buffer – typically TAE (Tris-Acetate-EDTA) or TBE (Tris-Borate-EDTA).
Loading dye – contains:
Dense component (e.g., glycerol) to ensure sample sinks in wells.
Tracking dyes to monitor sample migration during the run.
DNA samples – such as PCR products, plasmids, or genomic DNA.
DNA ladder – molecular weight marker (e.g., 1 kb ladder) for fragment size estimation.
Staining agents:
Ethidium bromide (EtBr) – intercalates with DNA and fluoresces under UV light.
Alternatives – safer dyes like SYBR Safe, GelRed, etc.
Glassware & Consumables
Beaker (250–500 ml) – for gel preparation.
Measuring cylinder – for buffer measurement.
Micropipettes (2–10 µL, 10–100 µL) – for accurate loading.
Sterile pipette tips – to prevent contamination.
Aluminum foil – used to protect light-sensitive dyes like EtBr from degradation.
Optional Dyes for Post-Staining (If Not Pre-stained)
Ethidium bromide soaking – post-run gel staining method.
1-anilino-8-naphthalene sulphonate – alternative extrinsic fluor for DNA detection.
Safety Precautions in Agarose Gel Electrophoresis
Ethidium Bromide Handling (Mutagenic Substance):
Always wear nitrile gloves when handling EtBr.
Dispose of gels and buffers containing EtBr in designated biohazard containers.
Avoid contact with skin and surfaces.
Agarose Powder Safety:
Handle in a well-ventilated area to prevent inhalation.
Do not shake, tap, or blow into the container to avoid airborne particles.
Handling Hot Agarose Gel:
Use heat-resistant gloves to prevent burns.
Pour hot agarose slowly to avoid splashing or skin contact.
UV Light Exposure:
Wear UV protective goggles or use a UV shield during gel visualization.
Never look directly into UV light.
Electrical Safety:
Ensure all cables and connections are dry before use.
Close the lid securely before turning on the power.
Switch off the power supply before opening the electrophoresis chamber.
Steps Involved in Agarose Gel Electrophoresis
1. Preparation of Agarose Gel Solution
Weigh the desired amount of agarose powder based on required concentration (typically 0.2%–3% w/v).
Mix agarose powder with TBE or TAE buffer in a heatproof beaker.
Heat the mixture using a microwave or heating plate until the agarose is fully dissolved (solution becomes clear).
2. Adjusting Gel Concentration
Low agarose concentration (e.g., 0.7%) → separates large DNA fragments.
High agarose concentration (e.g., 2%) → separates small DNA fragments.
3. Staining the Gel
Allow the solution to cool to ~60°C before adding ethidium bromide (final concentration: 0.5 µg/mL) or a safer alternative (e.g., SYBR Safe).
Mix gently to distribute the stain evenly.
4. Casting the Gel
Pour the warm agarose solution into a gel casting tray fitted with combs to form sample wells.
Let the gel solidify at room temperature (approx. 20–30 minutes).
Carefully remove the combs without damaging the wells.
5. Setting Up the Electrophoresis System
Place the solidified gel (in tray) into the electrophoresis chamber.
Pour fresh electrophoresis buffer over the gel until it is completely submerged.
6. Sample Loading
Mix DNA samples with loading dye (e.g., containing glycerol and tracking dyes like bromophenol blue).
Load the DNA ladder (molecular weight marker) and DNA samples into the wells using micropipettes.
7. Running the Gel
Place the lid on the chamber and connect the power supply.
Set appropriate voltage (typically 80–120 V).
Start the run; bubbles at electrodes confirm current is flowing.
DNA fragments migrate from negative (cathode) to positive (anode) due to the phosphate backbone.
8. Monitoring Progress
Tracking dyes (e.g., bromophenol blue) move through the gel and indicate how far the run has progressed.
Stop the run when dyes approach the gel’s bottom edge.
9. Visualization of DNA
Carefully remove the gel and place it on a UV transilluminator.
View the fluorescent DNA bands under UV light, comparing them with the DNA ladder for size estimation.
Applications of Agarose Gel Electrophoresis
Agarose gel electrophoresis is used for the separation of DNA, RNA, or proteins based on size and charge.
It helps in the estimation of DNA fragment sizes using DNA ladders as reference.
Commonly used to analyze PCR products, which is important in genetic diagnosis and DNA fingerprinting.
Used for separating digested genomic DNA before performing Southern blotting, and RNA before Northern blotting.
Employed in restriction mapping to study the pattern of DNA fragments after restriction enzyme digestion.
Useful for resolving circular DNA molecules with different supercoiling topologies.
Can distinguish between DNA fragments that differ due to DNA synthesis or chemical modifications.
Allows for the purification of specific DNA fragments by excising bands from the gel, which is essential for techniques such as cloning, sequencing, and further molecular manipulation.
Advantages of Agarose Gel Electrophoresis
Requires only agarose without the need for polymerization catalysts.
Simple and quick to prepare.
Gels are easily poured and handled.
Non-denaturing, preserving the native structure of DNA/RNA.
Samples can be recovered from the gel for further use.
Disadvantages of Agarose Gel Electrophoresis
Gels may melt during electrophoresis if overheating occurs.
Buffer can become exhausted, affecting separation quality.
Different DNA conformations (e.g., supercoiled, nicked, linear) may migrate unpredictably, complicating interpretation.
References
Semantics Scholar. (n.d.). Agarose Gel Electrophoresis – Principles and Applications. Retrieved from https://pdfs.semanticscholar.org/36cf/d4ada922c44d233b6ebfa2af2c956c92e4ec.pdf
Scarr, P. (n.d.). Gel Electrophoresis: Principles and Practice. Memorial University of Newfoundland. Retrieved from https://www.mun.ca/biology/scarr/Gel_Electrophoresis.html
Western Oregon University. (n.d.). Gel Electrophoresis Lab Manual. Retrieved from https://www.wou.edu/las/physci/ch462/Gel%20Electrophoresis.pdf
Wikipedia. (n.d.). Agarose Gel Electrophoresis. Retrieved from https://en.wikipedia.org/wiki/Agarose_gel_electrophoresis
Michigan State University. (2009). Plant Tissue Electrophoresis Lecture Notes. Retrieved from https://msu.edu/course/css/451/Lecture/PT-electrophoresis%20(2009).pdf
University of Macau Library. (n.d.). Electrophoresis Techniques E-Book. Retrieved from http://library.umac.mo/ebooks/b28050459.pdf
