The principle has dual functions of "molecular sieve" and "electrophoresis".

Agarose gel has a network structure, and the molecules of matter will be resisted when they pass through, and the macromolecules will be greatly resisted when they surge. Therefore, in gel electrophoresis, the separation of charged particles depends not only on the nature and quantity of net charges, but also on the size of molecules, which greatly improves the resolution. However, due to its large pore size, its molecular sieve effect is negligible for most protein, and it is now widely used in nucleic acid research. DNA molecules have charge effect and molecular sieve effect when swimming in agarose gel. DNA molecules are negatively charged in a pH solution above the isoelectric point, and move to the positive electrode in an electric field. Because of the repetitive structure of sugar-phosphate skeleton, the same number of double-stranded DNA has almost the same net charge, so they can move towards the positive electrode at the same rate.

Nucleic acid molecules are amphoteric dissociative molecules. In the electrophoresis buffer above its isoelectric point, their bases are inseparable, while all phosphate groups are dissociated, so the nucleic acid molecules are negatively charged and migrate to the positive electrode during electrophoresis. That is to say-there is phosphate on DNA, which is negatively charged under normal circumstances, so it is bound to move in the positive direction.

1, preparing and compounding glue

Second, the sample

1, run according to the amount you want. For example: 10μl sample, 4 and 6μl Mark, and carefully add them into the sample tank with a micro pipette.

Carefully add it into the sample tank with a micro-pipette gun, and replace the gun head after each sample is added to prevent mutual pollution. Pay attention to the careful operation when loading the sample to avoid damaging the gel or piercing the gel at the bottom of the sample tank.

Third, electrophoresis

1, after adding the sample, close the electrophoresis tank cover and turn on the power immediately. The control voltage is kept at 1 10 V, and the current is above 40 mA. Transverse pressure running

2. When the band moves to about 2 cm from the gel front (about 40 min), stop electrophoresis.

3. Turn on the computer and observation instrument, and take photos and observe under ultraviolet light.

Note: PCR products can be placed in the refrigerator at 4℃ after running.

Remarks:

1, gel thickness and pore size

Generally speaking, thicker gels generate more heat during operation, which may lead to the diffusion of bands. Due to the high background of gel dyeing or the time required for gel dyeing and/or decoloration (such as dyeing after electrophoresis), poor visualization may occur. For agarose gel, the thickness is preferably 3-4 mm, and gel with a thickness of more than 5 mm is not recommended. The thickness of polyacrylamide gel is determined by the gasket provided by the manufacturer for gel pouring, and the most common ones are 0.75 mm,1.0 mm,1.5 mm.

The pore size determined by the shape of the rubber comb not only affects the sample loading, but also affects the resolution of the strip. Although the larger hole can accommodate the larger sample load, it will also produce thick stripes, which will reduce the resolution of stripes and produce stains. However, the long and narrow hole can accommodate a small sample, but it can provide clearer bands to obtain better resolution. Reducing the injection volume of high-density samples can provide higher intensity bands.

2, run electrophoresis time

The electrophoresis time is short, and the DNA bands of different length fragments have not been separated, so only the main band with the highest DNA content can be seen. However, the electrophoresis time is long, and the DNA bands of different length fragments have been separated, and the DNA bands of different length fragments can be clearly seen.

3. The DNA band that ran out was blurred.