Agarose Gel Electrophoresis Detection of Plasmids

Agarose gel electrophoresis is an electrophoresis method that uses agar or agarose as a supporting medium. For samples with relatively large molecular weights, such as macromolecular nucleic acids, viruses, etc., agarose gels with larger pore sizes can generally be used for electrophoretic separation. Agarose gel can distinguish DNA fragments that differ by 100 bp. Although its resolution is lower than polyacrylamide gel, it is easy to prepare and has a wide separation range, and is especially suitable for separating large fragments of DNA.

Principle

Agarose gel electrophoresis is a routine method for the separation and identification of nucleic acids in genetic engineering laboratories. Nucleic acid is an amphoteric electrolyte with an isoelectric point of pH 2-2.5. In a conventional electrophoresis buffer (pH about 8.5), nucleic acid molecules are negatively charged and move toward the positive electrode in the electric field. When nucleic acid molecules swim in agarose gel, they have charge effects and molecular sieve effects, but mainly the molecular sieve effect. Therefore, the mobility of nucleic acid molecules is determined by the following factors.

• The size of DNA molecules. The migration rate of linear double-stranded DNA molecules in a certain concentration of agarose gel is inversely proportional to the logarithm of the relative molecular mass of the DNA. The larger the molecule, the greater the resistance and the harder it is to move in the gel pores, so it migrates slower.
• Conformation of DNA molecules. When the DNA molecule is in different conformations, the distance it moves in the electric field is not only related to the relative molecular mass, but also to its own conformation. Linear, open circular and supercoiled plasmid DNA with the same relative molecular mass move at different speeds in agarose gels. Supercoiled DNA moves the fastest, while open circular DNA moves the slowest. If several DNA bands are found on the gel during electrophoresis to determine the purity of the plasmid, and it is difficult to determine whether it is caused by different conformations of the plasmid DNA or the inclusion of other DNA, the DNA bands can be recovered one by one from the agarose gel. Use the same restriction endonuclease for hydrolysis and then electrophoresis. If the same DNA map appears on the gel, it is the same DNA.
• Power supply voltage. At low voltages, the migration rate of linear DNA fragments is proportional to the applied voltage. However, as the electric field strength increases, the mobility of DNA fragments with different relative molecular masses will increase at different rates. The larger the fragment, the greater the increase in mobility caused by the increase in field strength. Therefore, as the voltage increases, the effective separation range of the agarose gel will decrease. To achieve maximum resolution of DNA fragments larger than 2 kb, the applied voltage must not exceed 5 V/cm.
• Effect of ionic strength. The composition of the electrophoresis buffer and its ionic strength affect the electrophoretic mobility of DNA. When there are no ions (such as misusing distilled water to prepare gel), the conductivity is minimal and DNA hardly moves. In buffers with high ionic strength (such as accidentally adding 10× electrophoresis buffer), the conductivity is very high and heat is obviously generated. In severe cases, it may cause gel melting or DNA denaturation.

Ethidium bromide (EB) can be inserted into DNA molecules to form complexes. EB can emit fluorescence under the irradiation of ultraviolet light with a wavelength of 254 nm, and the intensity of fluorescence is proportional to the content of nucleic acid. If a standard sample with a known concentration is used as an electrophoresis control, the concentration of the sample to be tested can be estimated. Because ethidium bromide is suspected of causing cancer, safe dyes such as Sybergreen have also been developed.

Conventional horizontal agarose gel electrophoresis is suitable for the separation and identification of DNA and RNA, but agarose electrophoresis denatured by formaldehyde is more suitable for the separation and identification of RNA and Northern blot. Because the denatured RNA is a single strand, its swimming speed is the same as the relative molecular mass of DNA of the same size, so the size of the RNA molecule can be measured. Moreover, the stained bands are sharper and more firmly bound to the nitrocellulose membrane, enabling efficient hybridization with radioactive or non-radioactive labeled probes.

Procedures

1. Prepare 1% agarose gel

Prepare an agarose solution of appropriate concentration and heat it in a microwave until the agarose is completely melted. The concentration of agarose in the gel is determined based on the size of the DNA molecules to be separated. Please refer to Table 1.

Table 1 DNA separation range of standard agarose gels with different concentrations

2. Preparation of rubber plate

Take the inner glass tank (gel making tank) in the electrophoresis tank, wash it, dry it, and put in the gel making glass plate. Use transparent tape to seal the edges of the glass plate and the inner groove to form a mold. Place the inner groove in a horizontal position and place the comb in a fixed position. Mix the agarose gel solution that has been cooled to about 65°C and pour it carefully onto the glass plate in the inner tank. Let the gel slowly spread until a uniform ge layer forms on the entire surface of the glass plate. Let it stand at room temperature until the gel is completely solidified. Pull the comb gently vertically, remove the tape, and put the gel and inner tank into the electrophoresis tank. Add 1×TAE electrophoresis buffer until the gel plate is covered.

3. Add sample

Mix the DNA sample and loading buffer on the spotting plate. The final dilution factor of the loading buffer should be no less than 1×. Use a 10 µL micropipette to add the samples to the sample slots of the gel plate. After each sample is added, a sample adding head should be replaced to prevent contamination. Do not damage the gel surface around the sample hole when adding samples. (Note: Please note the order of adding samples before adding samples).

4. Electrophoresis

After adding the sample, the gel plate was immediately energized for electrophoresis at a voltage of 60-100 V. The sample moved from the negative electrode (black) to the positive electrode (red). As the voltage increases, the effective separation range of the agarose gel decreases. Stop the electrophoresis when the bromophenol blue moves to about 1 cm from the bottom edge of the gel plate.

5. Dyeing

After electrophoresis, take out the gel, stain it with 1×TAE solution containing 0.5 µg/mL ethidium bromide for about 20 min, and then rinse with water for 10 min.

6. Observe

When observed under ultraviolet light, the presence of DNA shows a red fluorescent band, which is photographed and preserved using a gel imaging system.

Note

• EB is a strong mutagen and is suspected of causing cancer. Wear gloves and masks during operation and be careful to separate experimental equipment contaminated with EB from those not contaminated with EB during the experiment to prevent EB cross-contamination.
• If the microwave heating time is too long, the agarose solution will become overheated or boil violently. The shortest heating time required until all agarose particles are completely dissolved should be chosen.
• There are three forms of plasmid DNA: covalently closed circular DNA, which often exists in a supercoiled form; open circular DNA, in which one of the two strands of plasmid DNA is broken at one or more places, so it can rotate freely to eliminate tension and form a relaxed circular molecule; linear DNA, which is caused by the two strands of plasmid DNA breaking at the same place. Therefore, three swimming bands may appear in the results of plasmid DNA electrophoresis, and their swimming speeds are: covalently closed DNA > linear DNA > open circular DNA.

Related Services

• Plant Genetic Engineering
• Detection of Plant Nematodes by Two-Dimensional Gel Analyses
• Gene Expression Profiling
• Northern Blot
• qPCR
• Gene Copy Number Analysis
• The Southern Blot Assay in Plants