Principles and advantages of agarose gel electrophoresis
Agarose gel electrophoresis is an electrophoretic method using agar or agarose as a supporting medium. For samples with larger molecular weights, such as macromolecular nucleic acids, viruses, etc., agarose gel with larger pore size can generally be used for electrophoretic separation.
Agarose Precast Gel Set
Principle of agarose gel electrophoresis:
Agarose gel electrophoresis is an electrophoretic method using agarose as a supporting medium. The main difference between its analysis principle and other support electrophoresis is that it has the dual functions of "molecular sieve" and "electrophoresis".
The agarose gel has a network structure, and the substance molecules are subjected to resistance when passing through, and the macromolecular substances are subject to large resistance when surging, so in gel electrophoresis, the separation of charged particles not only depends on the nature and number of net charges, but also It also depends on the size of the molecule, which greatly improves the resolving power. However, because its pore size is too large compared to proteins, the molecular sieve effect is insignificant for most proteins, and is now widely used in the study of nucleic acids.
Proteins and nucleic acids will have different electric charges according to different pHs and different forces in the electric field, so they run at different speeds, and they can be separated according to this principle. The pH of the electrophoresis buffer is between 6-9, and the ionic strength of 0.02-0.05 is the most suitable. 1% agarose is commonly used as a support for electrophoresis. Agarose gel can distinguish DNA fragments about 100bp apart. Although its resolution is lower than polyacrylamide gel, it is easy to prepare and has a wide separation range. Ordinary agarose gels can separate DNA in the range of 0.2-20kb. Using pulse electrophoresis, DNA fragments up to 10^7bp can be separated.
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 toward the positive electrode in the electric field. Due to the repetitive structure of the sugar-phosphate backbone, the same amount of double-stranded DNA has almost the same amount of net charge, so they can move toward the positive electrode at the same rate.
Advantages of agarose gel electrophoresis:
1. Agarose gel is a matrix with a large number of tiny pores, and its pore size depends on its concentration. The pore size of 0.075 agarose is 800nm, the pore size of 0.16 is 500nm, and the pore size of 1% agarose is 150nm. This is the concentration of commonly used agarose gel. It can separate about 0.1-60kb of DNA. Epidemic fixation, immunoelectrophoresis and micro preparation
2. Agarose has high mechanical strength, allowing it to be used at a concentration of 1% or lower; and only weakly binds to other organisms
3. Agarose is non-toxic, free radical polymerization will not occur during the solidification process of agarose gum, no catalyst is needed
4. Agarose gum has thermal reversibility, low melting point is easy to recover samples, and can be used for the preparation of temperature sensitive materials
5. Easy to store, it is an ideal material for highly sensitive autoradiography
Agarose prefabricated gels are essentially the same as ordinary agarose gels, except that the gels are prepared in advance in large quantities and pre-stained with nucleic acid dyes in advance, eliminating the time and purchase of a large amount of gel preparation when performing nucleic acid electrophoresis. And the steps of using nucleic acid dyes, generally need to be equipped with reagents such as agarose, nucleic acid dyes, electrophoresis solution and loading buffer. The preparation work is tedious and time-consuming and costly, and one kit of pre-gel agarose electrophoresis kit is all solved. This kit can save you about an hour of experiment time.
Electrophoresis generally needs to go through four steps: preparing electrophoresis solution, spotting, electrophoresis, and cleaning the electrophoresis tank. The entire process is time-consuming and labor-intensive. The agarose precast gel electrophoresis kit is equipped with a high-pressure fast electrophoresis solution 15mL, which improves the efficiency of electrophoresis while saving time and procurement costs.
In chemiluminescence analysis, the luminescence intensity of acridine ester is influenced by various factors, such as reaction medium, temperature, time, and excitation light source energy. To achieve good detection results, it is necessary to comprehensively consider and optimize these factors. Meanwhile, attention should be paid to controlling and standardizing experimental conditions to ensure accurate and reliable results. Thoroughly studying these influencing factors will help promote the development of chemiluminescence analysis methods.