Agarose gel electrophoresis in dna influence migration speed:

1, the molecular size and configuration of DNA

Different configurations of DNA moving speed order: valence-dependent closed circular DNA (covalently closed circular,cccDNA)> linear DNA> open double-stranded circular DNA. The migration rate of linear double-stranded DNA molecules in a certain concentration of agarose gel is inversely proportional to the logarithm of the molecular weight of DNA, and the larger the molecule is, the greater the resistance, and the more difficult it is to creep through the pores of the gel.

Thus, the slower the migration. When the concentration of agarose is too high, cyclic DNA (generally spherical) cannot enter the gel, and the relative mobility is 0 (Rm=0), while linear double-stranded DNA of the same size (rigid rods) can move forward in the direction of the long-axis (Rm>0), which shows that the relative mobility of the three configurations is mainly dependent on the concentration of the gel.

2, agarose concentration

A given size of the linear DNA molecule, its migration rate in different concentrations of agarose gel varies. The logarithm of the DNA electrophoretic mobility is linearly related to the gel concentration. The choice of gel concentration depends on the size of the DNA molecules. The gel concentration required for separation of DNA segments smaller than 0.5 kb is 1.2-1.5%, for separation of DNA molecules larger than 10 kb is 0.3-0.7%, and for DNA segments in between, 0.8-1.0%.

3, the conformation of the DNA molecule

When the DNA molecule is in a different conformation, it is not only related to the molecular weight of the molecule, but also related to its own conformation of the distance it moves in the electric field. Linear, open-loop and superhelical DNAs of the same molecular weight move differently in an agarose gel, with superhelical DNA moving the fastest and linear double-stranded DNA moving the slowest.

These molecules have the same molecular weight, but they move at different speeds in an agarose gel. If several DNA bands are found on the gel during electrophoresis to identify the purity of the plasmid, and it is difficult to determine whether they are caused by different conformations of the plasmid DNA or by the presence of other DNAs, the DNA bands can be recovered from the agarose gel one by one and hydrolyzed with the same kind of restriction endonuclease, then electrophoresed, and if the same DNA pattern appears on the gel, then it is the same kind of DNA.

4.Supply voltage

The results of the experimental conditions for the separation of large molecules of DNA on agarose gel show that the separation effect is better at low concentration and low voltage. The electrophoretic mobility of linear DNA molecules is proportional to the voltage used under low voltage conditions.

However, the mobility of DNA segments with different molecular weights will increase with different magnitudes when the electric field strength is increased. The larger the segment, the greater the increase in mobility due to the increase in field strength, and therefore the effective separation range of the agarose gel will be narrowed by the increase in voltage. To maximize the resolution of DNA segments larger than 2 kb, the field strength should not be higher than 5 V/cm.

5. Presence of Embedding Dye

The fluorescent dye ethidium bromide is used for the detection of DNA in agarose gels, and the dye is embedded in the stacked base pairs and elongates the threads and notched loops of the DNA, which makes them more rigid and decreases the mobility of the threads. 15 percent.

6. Effect of ionic strength

The composition of the electrophoresis buffer and its ionic strength affect the electrophoretic mobility of DNA. When no ions are present (e.g., when the gel is mistakenly prepared with distilled water), the conductivity is minimal and the DNA is almost immobile. In a buffer with high ionic strength (e.g., when 10× electrophoresis buffer is mistakenly added), the conductivity is very high and the heat generation is obvious, which can cause the gel to melt or the DNA to be denatured in severe cases. For natural double-stranded DNA, several electrophoresis buffers are commonly used, such as TAE [containing EDTA (pH 8.0) and Tris-acetic acid], TBE (Tris-boric acid and EDTA), TPE (Tris-phosphoric acid and EDTA), which are usually prepared as concentrated master batches and stored at room temperature.