Nucleic acid electrophoresis is an important means of nucleic acid research, which is an indispensable part of nucleic acid probe, nucleic acid amplification and sequence analysis.
Nucleic acid electrophoresis is usually carried out in agarose gel or polyacrylamide gel.
Different concentration of agarose and polyacrylamide can form gel with different mesh size of molecular sieve, which can be used to separate nucleic acid fragments with different molecular weight.
1.Wash the glue making mould and comb with distilled water, put them on the glue making plate, close the edge of mould and frame the comb.
2.Prepare agarose gel of appropriate concentration with gel buffer according to the size of DNA fragment to be separated: accurately weigh the dry agarose powder, add it into the triangular flask for gel preparation, and quantitatively add the electrophoresis buffer (generally 20-30 ml).
3.Put it into the microwave oven for heating and melting. Cool for a moment, add a drop of fluorescent dye, rotate it gently to mix the gel solution well, pour it into the electrophoresis tank, and let it solidify.
4.At room temperature, the gel is completely condensed after 30~45 minutes. Carefully pull out the comb and place the gel in the electrophoresis tank.
5.Pour the electrophoresis buffer into the electrophoresis tank, the amount of which should be 1mm from the unglued surface. If there are bubbles in the sample hole, try to remove them.
6.Add 10 × volume of load ing buffer into DNA sample, mix well, and then slowly add the sample mixture into the immersed gel sample hole with gun.
7.Turn on the power, red is the positive pole, black is the negative pole. Remember that the DNA sample swims from the negative pole to the positive pole (the end near the sample adding hole is negative). Generally 60-100v, electrophoresis for 20-40min.
8.Determine whether to terminate electrophoresis according to the position of indicator stroke.
9.After electrophoresis, turn off the power supply, observe the electrophoresis band and its position on the gel imager, and compare the size of the amplified product with the molecular weight standard marker of nucleic acid.
| Optimum concentration range of agarose gel and linear DNA | |
| Agarose gel concentration | The best resolution range of linear DNA(bp) |
| 0.5% | 1,000～30,000 |
| 0.7% | 800～12,000 |
| 1.0% | 500～10,000 |
| 1.2% | 400～7,000 |
| 1.5% | 200～3,000 |
| 2.0% | 50～2,000 |
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