Experimental operation of agarose gel electrophoresis-Electrophoretic Materials

Agarose gel electrophoresis is a common technique for the separation and purification of DNA fragments. The DNA sample is added to the sample hole of a porous support medium (agarose gel) containing electrolyte and placed on the electrostatic field. Due to the presence of negatively charged phosphate residues on both sides of the double helix skeleton of DNA molecules, they move towards the positive electrode in an electric field. At a certain electric field strength, The migration speed of DNA molecules depends on the molecular sieve effect. 

The swimming speed of DNA fragments with different relative molecular weights varies, so they can be separated based on the size of the DNA molecule. Gel electrophoresis can not only separate DNA with different molecular weights, but also separate DNA molecules with the same relative molecular weight but different configurations. During the electrophoresis process, detection can be obtained by using tracer dyes or relative molecular weight standard reference materials to electrophorese together with the sample. The relative molecular weight standard reference material can provide a standard for determining the size of DNA fragments. A small amount of ethidium bromide (EB) is added to the gel, and its molecule can be inserted between the bases of DNA to form a complex. Under the irradiation of 254-365nm ultraviolet light, it shows orange red fluorescence, so it can also detect the separated DNA.

The general agarose gel electrophoresis is suitable for DNA fragments in the size range of 0.2kb-50kb. This experiment introduces the preparation of agarose gel and the application of agarose gel electrophoresis in the separation of DNA fragments.

Horizontal electrophoresis tank, electrophoresis instrument, gel imaging analysis system, microwave oven, micropipette, transparent tape, spot template or parafilm 100 ml or 250 ml conical flask, graduated cylinder, suction head, etc.

Preparation of 50 × TAE buffer solution: 2 mol/L Tris acetic acid, 0.05 mol/L EDTA （ pH 8.0 ）

▲ Prepare 1000 ml

Tris 242 g

Iced acetic acid 57.1 ml

0.5 mol/L EDTA 100 ml

Add 600 ml of deionized water and stir to dissolve, then bring the solution to a volume of 1 L. High temperature and high pressure sterilization, stored at room temperature.

Preparation of 1 × TAE buffer:

Weigh 20 ml of 50 × TAE buffer and add 980 ml of deionized water.

Ethidium bromide storage solution: 10 mg/ml Ethidium Bromide

▲ Preparation: 100 ml

Weigh 1 g of ethidium bromide and place it in a 100 ml beaker. Add 80 ml of deionized water and stir to dissolve. After diluting the solution to 100 ml, transfer it to a brown bottle

. Store at room temperature.

6 x Sample buffer: 0.25% bromophenol blue, 0.25% xylene blue FF, 30% glycerol.

▲ Preparation: 10 ml

Bromophenol blue 25 mg

Xylene blue FF 25 mg

Glycerol 3 ml

Dissolve in 6 × TAE buffer to 10 ml and divide into 1 ml tubes- Store at 20 ℃.

Other reagents: DNA samples DNA Ladder 、 Agarose

1. Prepare 1% agarose gel (70ml for large gel and 50ml for small gel): weigh 0.7g (0.5g) agarose into a conical flask, add 70ml (50ml) 1 × TAE, and turn the beaker upside down. Heat and boil in microwave oven for 3 times until agarose is completely melted, shake well, and then 1.0% agarose gel liquid is formed.

2. Preparation of adhesive plate: Clean the organic glass inner tank (adhesive tank) in the electrophoresis tank, air dry it, and place it in the adhesive glass plate. Take transparent tape to seal the edges of the glass plate and the inner groove at both ends, forming a mold. Place the inner groove in a horizontal position and place the comb in a fixed position. Mix the agarose gel solution cooled to about 65 ℃ and carefully pour it onto the glass plate of the inner tank, so that the glue solution develops slowly until a uniform glue layer is formed on the surface of the whole glass plate. Let it stand at room temperature until the gel is completely solidified, pull out the comb vertically, take off the tape, and put the gel and inner tank into the electrophoresis tank. Add 1 x TAE electrophoresis buffer until it passes through the gel plate.

3. Sample adding: mix DNA sample and sample loading buffer on the spot sample plate or parafilm, and the final dilution ratio of the sample loading buffer should not be less than 1X. Use a 10 ul micropipette to add the sample into the sample tank of the rubber plate respectively. 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 during sample adding. (Note: Before adding samples, make a note of the order in which they are added.).

4. Electrophoresis: After the sample is added, the gel plate is immediately electrified for electrophoresis. The voltage is 60-100V, and the sample moves from negative (black) to positive (red). The effective separation range of agarose gel decreased with the increase of voltage. Stop electrophoresis when bromophenol blue moves to a distance of about 1cm from the bottom edge of the gel plate.

5. After electrophoresis, take out the gel, dye it with 1 × TAE solution containing 0.5 ug/ml ethidium bromide for about 20 min, and rinse it with clean water for 10 min.

6. Observation and photography: when observed under the ultraviolet light, the presence of DNA will show red fluorescent bands, which will be photographed and saved by the gel imaging system.

1. When preparing agarose, it should be completely melted before making the gel.

2. Agarose gel is easy to be broken and should be operated gently.

3.During electrophoresis, attention should be paid to the power supply circuit to prevent electric shock.

4. Ethyl bromide has carcinogenic effects, and latex or disposable plastic gloves should be worn during preparation and use. And use it in a specialized laboratory.

5. Ultraviolet radiation has a harmful effect on the human body, so it is not advisable to turn on the lights for too long and pay attention to protection.

6. Blurred DNA band shape: too much DNA added; Voltage too high; There are bubbles in the gel.

7.There are three forms of plasmid DNA:

  ① Covalent closed loop DNA (cccDNA) often exists in a supercoiled form;

  ② Open loop DNA (ocDNA),

One of the two strands of this plasmid DNA breaks at one or more points, allowing it to rotate freely to eliminate tension and form a relaxed circular molecule;

  ③ Linear DNA is caused by two strands of plasmid DNA breaking at the same location. Therefore, there may be three swimming bands in the results of plasmid DNA electrophoresis, with a swimming speed of cccDNA>linear DNA>ocDNA.