1, DNA preparation
⑴ It is suitable for separating large fragments of DNA.
Agarose gel can distinguish DNA fragments with a difference of about 100bp. Although its resolution is lower than that of polyacrylamide gel, it is easy to prepare and has a wide separation range, which is especially suitable for separating large fragments of DNA. The range of DNA separation by common agarose gel is 0.2-20kb, and DNA fragments as high as/10 7bp can be separated by pulse electrophoresis.
In the experiment of extracting total DNA from silage, agarose gel electrophoresis and ultraviolet absorption were used. The extracted DNA has high purity and can be directly used for downstream molecular manipulation. The extraction method has high sensitivity and can fully reflect the original appearance of microorganisms in the sample [1].
In the experiment of "CTAB combined with DNA gel recovery kit to extract DNA from edible fungi", researchers used two methods to extract DNA: one is CTAB method (control method). Second, the combination method of CTAB and DNA gel recovery kit (agarose gel DNA kit) (combination method). The experimental results show that compared with the control method, the electrophoresis bands of DNA samples extracted by the combined method are more regular and clear, and the DNA digestion efficiency is obviously higher than that by the control method. This shows that the combined method can effectively separate pure DNA from polysaccharide-rich materials [2].
⑵ Macromolecular DNA can be extracted.
In the experiment of "Preparation and Enzymatic Digestion of HMW DNA from Burkholderia cepacia", it was mentioned that the key to construct large fragment genome library is to obtain high molecular weight genome DNA[3]. However, using mature commercial kits to extract genomic DNA can only obtain DNA with a size of about 20kb. Enzymatic extraction requires multiple extractions to obtain pure DNA, but it is easy to break the genome and it is difficult to obtain genomic DNA larger than 300kb. Neither of the two methods can achieve the goal, but after unremitting research by researchers, a large enough DNA fragment can be obtained by using agarose gel to extract genomic DNA, which can completely meet the requirements of constructing cosmid genome library. In this process, researchers chose low-melting agarose or ordinary-melting agarose to prepare gel blocks, because low-melting agarose is expensive and gel blocks are easy to break during preparation and purification, and there is no difference between ordinary-melting agarose and low-melting agarose in genome DNA preparation.
1. PCR products were detected by agarose gel electrophoresis.
In the extraction of genomic DNA, the DNA was incubated, extracted and precipitated, washed twice with 70% ethanol, and then dissolved with appropriate amount of double distilled water. Then electrophoresis was carried out on 1.0% agarose gel, and the concentration of DNA solution was estimated with reference to 1kb DNA ladder. The specific test method is as follows: 2.0% agarose is used as gel, 6 microliters of AFLP—PCR products are mixed with 1 microliters of loading buffer, the electrophoresis buffer is 1×TBE, and the electrophoresis buffer is 120V for 50 minutes. After staining with EB solution, the samples were observed and photographed on a gel imaging system (ChampGel-3200) [4].
2. Application of agarose in other fields.
⑴ Application of agarose in immunodiffusion method.
Immunodiffusion method is to use agarose gel as diffusion medium, so that antigen and antibody can freely diffuse and meet in agarose gel, thus forming antigen-antibody complex. Because the molecular weight of this compound increases and aggregates, it will not continue to spread, forming a belt or linear precipitation belt visible to the naked eye. The precipitation zone of antigen-antibody complex is a specific semi-permeable barrier, which can prevent the antigen-antibody molecules with similar immunological properties from passing through, but allow those molecules with different properties to continue to spread, so that the precipitates formed by different antigens or antibodies have their own positions, so that the mixed system can be separated and identified.
Agarose gel is used as diffusion medium because a certain concentration of agarose gel has a porous network inside. Moreover, the pore size is very large, allowing macromolecular substances (molecular weight from hundreds of thousands to millions) to pass freely. Because most antigens and antibodies have molecular weights above 200,000, they can almost freely diffuse in agarose gel. Moreover, agarose gel has the advantages of good chemical stability, high water content, good transparency, convenient source and easy handling, and is the most ideal diffusion medium in immunoprecipitation detection technology.
In the two-way agar diffusion experiment, agar or agarose is also used as diffusion medium for the same reason as above.
⑵ The role of agarose in the synthesis of double-stranded DNA probes.
The synthesis methods of double-stranded DNA probes mainly include incision translation and random primer synthesis.
Notched translation method
There are several factors that affect the gap translation reaction: (a) The specific activity of the product depends on the specific activity of [α-32 P]dNTP and the degree of nucleotide substitution in the template. (The dosage of DNase ⅰ and the quality of DNA polymerase in Escherichia coli will affect the size of the product fragment. (c) Inhibitors such as agarose in DNA templates will inhibit the activity of enzymes, so carefully purified DNA should be used.
Random primer synthesis method
Random primer synthesis can also be carried out directly in low melting point agarose.
These are some applications of agarose gel in various fields of biology. However, due to the rapid development of biological research, agarose gel may be more widely used, and it will play its due role in biological research.
Development examples: Agar and agarose have special stabilizing effects and are easy to absorb water because of their special gel properties, especially their remarkable stability, hysteresis and hysteresis; It has been widely used in food, medicine, chemical industry, textile, national defense and other fields. According to incomplete statistics, there are more than 1000 uses of agar and agarose, which are called "new products in East Asia" internationally. In the food industry, it can be used to produce: crystal soft candy, shaped soft candy, aquatic products, canned meat, fruit juice drinks, pulp drinks, rice wine drinks, milk drinks, fine products and milk cakes.