Agarose gel electrophoresis is a widely used and reliable method for separating and analyzing DNA fragments. This technique is known for its simplicity, speed, and effectiveness in resolving DNA pieces that may be difficult to distinguish using other methods like density gradient centrifugation. When stained with a low concentration of the fluorescent dye ethidium bromide (EB), which is available from Deyuan International (Cat# LY5009), it becomes possible to visualize DNA bands as small as 1–10 ng under UV light. This allows researchers to easily locate and identify DNA fragments within the gel. Additionally, specific DNA bands can be excised from the gel after electrophoresis for further cloning or analysis.
The agarose gel system is highly versatile, capable of separating DNA fragments ranging from 200 base pairs up to nearly 50 kilobases. The separation efficiency depends on the concentration of the agarose gel, which is adjusted based on the size of the DNA fragment being analyzed. Typically, gels with concentrations between 0.3–0.7% are suitable for larger DNA molecules (>10 kb), while gels with 1.2–1.5% agarose are better for smaller fragments (<0.5 kb). For DNA fragments in the intermediate range, a 0.8–1.0% gel is usually recommended.
To perform agarose gel electrophoresis, you will need the following materials: DNA samples, agarose, and appropriate buffers. Essential equipment includes a horizontal electrophoresis unit, power supply, benchtop centrifuge, water bath, micropipettes, microwave or hot plate, UV transilluminator, camera setup, and a photo holder. Reagents such as 5× TBE buffer (Deyuan International Cat# LY5010), 6× loading buffer (Cat# LY50013), and a stock solution of ethidium bromide (10 mg/ml) should also be prepared.
For gel preparation, start by diluting 5× TBE buffer to 0.5×. Weigh 0.4g of agarose and dissolve it in 50ml of the diluted buffer. Heat the mixture in a microwave or on a hot plate until fully melted, then cool to around 50–60°C before adding EB to a final concentration of 0.5 μg/ml. Pour the solution into a mold with a comb inserted, allowing it to solidify. Once set, carefully remove the comb and fill the tank with 0.5× TBE buffer.
When loading the sample, mix 10 μl of DNA with 2 μl of 6× loading buffer and gently pipette into the wells. Adjust the volume if needed, but ensure it doesn’t exceed the well capacity. During electrophoresis, maintain a voltage of 60–80V and a current above 40mA. Stop when the bromophenol blue marker has migrated about 2 cm from the front.
After electrophoresis, stain the gel in a 0.5 μg/ml EB solution for 20–25 minutes. Visualize the DNA bands under a 254 nm UV lamp, wearing protective eyewear. Use a camera with a red filter and close-up lens to capture the image, adjusting exposure time based on band intensity. This detailed procedure ensures accurate and reproducible results in DNA analysis.
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