Agarose gel electrophoresis is one of the most common electrophoresis techniques which is relatively simple and straightforward to perform but possesses great resolving power. The agarose gel consists of microscopic pores that act as a molecular sieve that separates molecules based on the charge, size, and shape.
Agarose gel electrophoresis is a powerful separation method frequently used to analyze DNA fragments generated by restriction enzymes, and it is a convenient analytical method for separating DNA fragments of varying sizes ranging from 100 bp to 25 kb. DNA fragments smaller than 100 bp are more effectively separated using polyacrylamide gel electrophoresis whereas pulse-field gel electrophoresis is used to separate DNA fragments larger than 25 kb. Agarose gel electrophoresis can also be used to separate other charged biomolecules such as RNA and proteins.
The separation medium is a gel made from agarose. Agarose is isolated from the seaweed genera Gelidium and Gracilaria and consists of repeated agarobiose (L- and D-galactose) subunits. During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel’s molecular sieving properties. In general, the higher the concentration of agarose, the smaller the pore size.
To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current is applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size.
Factors affecting the migration of DNA
The mobility of DNA molecules is inversely proportional to gel concentration. Higher percentage gels are sturdier and easier to handle but the mobility of molecules and staining will take longer because of the tighter matrix of the gel. The most common agarose gel concentration for separating dyes or DNA fragments is 0.8%. However, some experiments require agarose gels with a higher percentage, such as 1% or 1.5%.
Size of DNA molecule
The sieving properties of the agarose gel influence the rate at which a molecule migrates. The separation occurs because smaller molecules pass through the pores of the gel more easily than larger ones. If the size of the two fragments is similar or identical, they will migrate together in the gel.
Different forms of DNA move through the gel at different rates; DNA molecules having a more compact shape (e.g. plasmid DNA) moves faster through the gel compared with linear DNA fragments of the same size. The migration rate of linear fragments of DNA is inversely proportional to log 10 of their size in base pairs. This means that the smaller the linear fragment, the faster it migrates through the gel.
Mobility of DNA molecules is also affected by the applied voltage. Within a range, the higher the applied voltage, the faster the sample migration.
Preparation of Agarose gel matrix
The centerpiece of agarose gel electrophoresis is the horizontal gel electrophoresis apparatus. The gel is made by dissolving agarose powder in a boiling buffer solution.
The concentration of agarose in a gel depends on the sizes of the DNA fragments to be separated, with most gels ranging between 0.5%-2%. The solution is then cooled to approximately 55°C and poured into a casting tray which serves as a mold. A well-former template (often called a comb) is placed across the end of the casting tray to form wells when the gel solution solidifies.
After the gel solidifies, the gel is submerged in a buffer-filled electrophoresis chamber which contains a positive electrode (anode) at one end, and a negative electrode (cathode) at the other. The volume of the buffer should not be greater than 1/3 of the electrophoresis chamber. The most common gel running buffers are TAE (40 mM Tris-acetate, 1 mM EDTA) and TBE (45 mM Tris-borate, 1 mM EDTA).
Sample preparation and loading
Samples are prepared for electrophoresis by mixing them with loading dyes. Gel loading dye is typically made at 6X concentration (0.25% bromphenol blue, 0.25% xylene cyanol, 30% glycerol). Loading dyes used in gel electrophoresis serve three major purposes:
- add density to the sample, allowing it to sink into the gel.
- provide color and simplify the loading process.
- the dyes move at standard rates through the gel, allowing for the estimation of the distance that DNA fragments have migrated.
These samples are delivered to the sample wells with a clean micropipette (variable automatic micropipette is the preferred one).
Ethidium bromide can be added to the gel during this step or alternatively, the gel may also be stained after electrophoresis in running buffer containing 0.5 μg/ml EtBr for 15-30 min, followed by destaining in running buffer for an equal length of time.
Applying electric current and separating biomolecules
A direct current (D.C.) power source is connected to the electrophoresis apparatus and an electrical current is applied. Charged molecules in the sample enter the gel through the walls of the wells. Molecules having a net negative charge migrate towards the positive electrode (anode) while net positively charged molecules migrate towards the negative electrode (cathode). The buffer serves as a conductor of electricity and controls the pH, which is important to the charge and stability of biological molecules. Since DNA has a strong negative charge at neutral pH, it migrates through the gel towards the positive electrode during electrophoresis.
The bluish-purple dye allows for visual tracking of sample migration during electrophoresis. The gel is run until the dye has migrated to an appropriate distance.
The agarose gel will have to be post-stained after electrophoresis. The most commonly used stain for visualizing DNA is ethidium bromide (EtBr)*. Alternative stains for DNA in agarose gels include SYBR Gold, SYBR green, crystal violet, and methyl blue. The sensitivities of methylene blue and crystal violet are low compared with ethidium bromide. SYBR gold and SYBR green are highly sensitive but more expensive than EtBr.
EtBr works by intercalating itself in the DNA molecule in a concentration-dependent manner. When exposed to a short wave ultraviolet light source (transilluminator), electrons in the aromatic ring of the ethidium molecule are activated, which leads to the release of energy (light) as the electrons return to the ground state. This allows for an estimation of the amount of DNA in any particular DNA band based on its intensity.
*Ethidium bromide is a suspect mutagen and carcinogen so must be handled cautiously. It is a hazardous waste so must be disposed of according to strict local and/or state guidelines. Stains containing methylene blue are considered safer than ethidium bromide, but should still be handled and disposed with care.
The exact sizes of separated DNA fragments can be determined by plotting the log of the molecular weight for the different bands of a DNA standard (DNA ladder) against the distance traveled by each band. The DNA standard contains a mixture of DNA fragments of pre-determined sizes that can be compared against the unknown DNA samples.
DNA concentrations can be estimated by:
A. Taking absorbance at 260 nm.
At 260 nm, an absorbance (A) of 1 unit corresponds to a concentration of:
- 50 μg/ml for dsDNA
- 40 μg/ml for RNA
- 33 μg/ml for ssDNA
- 20-30 µg/ml for oligonucleotides
Although this method is quick and nondestructive and gives information about the purity of the sample (e.g., presence of protein or organic contaminants), reliable estimates are obtained only with concentrations of at least 1 μg/ml. Additionally, this method cannot distinguish between DNA and RNA.
B. Intensity of Ethidium Bromide Fluorescence:
The amount of DNA in a sample can be estimated from the intensity of ethidium bromide fluorescence (fluorescence emitted by ethidium bromide is proportional to the amount of DNA). The DNA quantity in an “unknown” solution can be estimated by comparing its level of fluorescence with the intensity of known amounts of DNA of similar size. This method is useful if a DNA sample is contaminated with other compounds that absorb in the UV range or is too dilute to measure at 260 nm.
References and further reading
- Agarose Gel Electrophoresis for the Separation of DNA Fragments. Pei Yun Lee, John Costumbrado, Chih-Yuan Hsu, Yong Hoon Kim J Vis Exp. 2012; (62): 3923.
- Principles and Practice of Agarose Gel Electrophoresis: EDVOTEK
- Agarose gel electrophoresis: Rice University
- Agarose gel electrophoresis of DNA – Principle, Protocol and Uses: Laboratoryinfo.com