Agarose gel electrophoresis (200 bp-5 kb)

Agarose is a commonly used medium for separating DNA molecules. It is a linear polysaccharide polymer that can form rigid pores. The size of the gel pores depends on the concentration of agarose. The higher the concentration, the smaller the pores and the stronger the ability to separate small molecular weight DNA. DNA is negatively charged under alkaline conditions, and moves to the positive electrode through the gel medium in the electric field. Different DNA molecular fragments have different swimming rates in the electric field due to their different molecules and configurations. Ethidium bromide (EB) can be embedded in DNA molecules to form EB-DNA complexes, which will fluoresce under ultraviolet light. The molecular weight and concentration of sample DNA^[1][2].

1. Glue preparation:

Select the appropriate gel solution concentration for preparation. For example, weigh 1g of agarose and place it in a conical flask, add 100 mL of 1×TAE or TBE dilution buffer, heat until the agarose is completely melted, and shake well to obtain a 1% agarose gel. When the uniformly dissolved glue solution is cooled to not too hot, add 2-3 μL of ethidium bromide (final concentration is about 0.5 μg/mL), and mix well.

2. Rubber sheet preparation:

Install the electrophoresis tank: wash the plexiglass electrophoresis gel bed, dry it, seal the openings at both ends with tape, place it on a horizontal workbench, and insert a sample comb.

Gel pouring: Gently pour the agarose solution cooled to 60°C on the horizontal plate of the electrophoresis tank. After the agarose gel solidifies, add enough electrophoresis buffer in the electrophoresis tank to cover the surface of the gel, and then pull out the comb.

3. Add sample:

Take an appropriate amount of sample and 6× loading buffer (the main components are 0.25% bromophenol blue, 0.25% xylene blue, 30% glycerol. The loading dye helps to track the distance traveled by the DNA sample, and can also allow the sample to sink into the gel.

Mix well (eg: 1 μL sample with 5 μl 6× loading buffer), then carefully add to the sample well with a micropipette. The sample volume can be appropriately adjusted according to the sample concentration.

If the DNA content is low, the sample volume can be increased according to the above ratio, but the total volume should not exceed the capacity of the sample tank (generally, 40 μL for small holes is the upper limit, and 200 μL for large holes is the upper limit. The details are related to the specifications of the film).

It is recommended to replace the tip of the pipette after adding a sample to prevent mutual contamination. Be careful when loading the sample to avoid damaging the gel or piercing the gel at the bottom of the sample tank.

4. Electrophoresis

After adding the sample, close the cover of the electrophoresis tank and turn on the power. Set the voltage to the desired level, typically 1 to 10 V/cm. When the band moves to about 2 cm from the front of the gel (about 40 min), stop the electrophoresis. Take pictures and observe under ultraviolet light. Ethidium bromide is a carcinogen, so be careful when handling it, and gloves must be worn.

1. The buffer used for electrophoresis and the buffer used for gel making must be the same.

2. When dissolving the agarose, it is necessary to ensure that the agarose is fully dissolved, otherwise the electrophoretic image will be blurred.

3. Pour the melted agarose, insert the gel comb, make sure there are no air bubbles under the comb, and remove all air bubbles on the surface of the agarose before the gel solidifies to prevent the electrophoretic image from being blurred by air bubbles.

4. When the gel is not used immediately, please wrap the gel with plastic wrap and store it at 4°C to prevent it from drying out. Generally, it can be stored for 2 to 5 days.