Agarose gel electrophoresis is the most commonly used method for isolating and analyzing DNA fragments. It is a straightforward and efficient technique that can separate DNA fragments that are difficult to distinguish using other methods like density gradient centrifugation. When stained with a low concentration of ethidium bromide (EB), a fluorescent intercalating dye, DNA bands as small as 1–10 ng can be visualized under ultraviolet light, allowing precise determination of the DNA fragment's position in the gel. Moreover, specific DNA bands can be excised from the gel after electrophoresis for further cloning or analysis.
This method is highly versatile, capable of separating a wide range of DNA fragment sizes, typically from around 200 base pairs up to nearly 50 kilobases. The separation efficiency depends on the agarose concentration, which can be adjusted based on the size of the DNA being analyzed. Agarose gels are usually run horizontally using a constant electric field, ensuring consistent and reliable results.
The electrophoretic mobility of DNA is logarithmically related to the agarose concentration. For DNA fragments smaller than 0.5 kb, a 1.2–1.5% agarose gel is recommended, while larger fragments (>10 kb) require a lower concentration, around 0.3–0.7%. For intermediate sizes, a 0.8–1.0% gel is ideal. This flexibility allows researchers to tailor the gel to their specific experimental needs.
**Materials Required:**
- DNA samples
- Agarose
- Electrophoresis buffers
- Ethidium bromide (EB) solution
**Equipment Needed:**
- Horizontal electrophoresis apparatus
- Power supply unit
- Benchtop high-speed centrifuge
- Constant temperature water bath
- Micropipettes
- Microwave or hot plate
- UV transilluminator
- Camera and photo holder
**Reagents Used:**
1. 5× TBE buffer (Deyuan International Cat# LY5010)
2. 6× loading buffer (Deyuan International Cat# LY50013)
3. EB stock solution: 10 mg/ml, stored in dark containers at room temperature
**Preparation of Agarose Gel:**
1. Dilute 5× TBE buffer by adding 20 ml of it to 180 ml of distilled water, making 200 ml of 0.5× TBE buffer.
2. Weigh 0.4 g of agarose and dissolve it in 50 ml of 0.5× TBE buffer in a 200 ml flask. Heat in a microwave or on a hot plate until fully melted. Shake occasionally during heating to ensure even dissolution. Cover the container to minimize evaporation.
3. Pour the molten agarose into a gel tray with a comb inserted, ensuring the comb teeth are about 1 mm above the bottom of the mold. Once cooled slightly (to 50–60°C), add 0.5 μg/ml EB to the gel before pouring.
4. Allow the gel to solidify completely, then carefully remove the comb. Fill the electrophoresis tank with 0.5× TBE buffer until it just covers the gel.
**Loading Samples:**
Mix 10 μl of the DNA sample with 2 μl of 6× loading buffer and load it into the wells using a micropipette. Avoid overloading the wells. Change pipette tips between samples to prevent cross-contamination.
**Electrophoresis:**
Apply a voltage of 60–80 V, maintaining a current above 40 mA. Stop the run when the bromophenol blue marker has migrated approximately 2 cm from the well.
**Staining and Visualization:**
After electrophoresis, stain the gel in a 0.5 μg/ml EB solution for 20–25 minutes. Visualize under a 254 nm UV lamp. DNA appears as orange-red fluorescent bands. Use protective eyewear when working under UV light. Photograph the gel using a camera equipped with a red filter and close-up lens, adjusting exposure time based on band intensity.
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