DNA agarose gel electrophoresis experimental principle and method steps
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September 17 00:04:23, 2022
First, the purpose of the experiment
Learn and master the principles and methods of DNA identification by agarose electrophoresis.
Second, the experimental principle
Agarose gel electrophoresis is a standard method for isolating, identifying and purifying DNA fragments. Agarose is a polysaccharide extracted from agar. It is hydrophilic, but not charged, and is a good electrophoresis support. DNA under negative conditions (pH 8.0 buffer) is negatively charged and moves through the gel medium to the positive electrode in an electric field. Different DNA molecule fragments have different mobility rates in the electric field due to different molecules and configurations. Ethidium bromide (EB) can be inserted into the base pair of DNA molecules to form a fluorescent complex. After ultraviolet irradiation, different zones can be separated to achieve separation, identification of molecular weight and screening of recombinants. Shanghai Chuangsai Technology provides 1239-45-8, ethidium bromide (EB), BR score 99%, commodity number: D16-1013683-1g, price 518 yuan.
Third, the experimental materials
DNA sample extracted in Experiment 14,
Fourth, appliances and drugs
Electrophoresis apparatus, electrophoresis tank, UV transflectometer, constant temperature water bath, microwave oven, micro-injector, tris, hydrochloric acid, sodium acetate, EDTA, agarose, bromophenol blue, ethidium bromide. Shanghai Chuangsai Technology provides 77-86-1, Tris, Tris>99%, molecular biology, commodity number: C84-3739-100g, price 65 yuan.
Fifth, the experimental steps
1, install the electrophoresis tank
The plexiglass electrophoresis gel bed was washed, air-dried, and the openings at both ends were sealed with tape, placed on a horizontal workbench, and the sample comb was inserted.
2. Preparation of agarose gel
Weigh agarose and dissolve it in running buffer. (According to 0.3-1.5% agarose content, 1-25 kb DNA with 1% gel, 20-100 kb DNA with 0.5% gel, 200-2000 bp The DNA is heated in a microwave or boiling water bath with a 1.5% gel to completely dissolve (do not heat to boiling), and shake out. Shanghai Chuangsai Technology provides 9012-36-6, agarose ITM, Agarose ITM, Gel Strength (1.5% gel) > 1,200 g/cm2, commodity number: C84-2678-5G, price 93 yuan.
3, filling glue
Gently pour the agarose solution cooled to 60 ° C into the electrophoresis tank horizontal plate.
4. After the agarose gel is solidified, add the electrophoresis buffer to the electrophoresis tank, and then pull out the comb.
5, sample loading
After the DNA sample was mixed with the loading buffer at 4:1, the mixture was added to the sample tank with a micropipette, and 10-20 μl was added per tank, and the sample order and the amount of the sample were recorded.
6, electrophoresis
Install the electrode lead. Connect one end of the sample hole to the negative pole and the other end to the positive pole. Turn on the power supply, adjust the voltage to 3-5V/cm, and electrophoresis for 1-3hr. When the bromophenol blue moves to the front edge of the gel 1-2cm, stop. Electrophoresis. Shanghai Chuangsai Technology provides JY-JX5 fast SSR electrophoresis tank, electrophoresis instrument, commodity number: C71-JY-JX5-set, the price is 1176 yuan.
7. Dyeing and observation
The gel was taken out and stained in a staining solution containing ethidium bromide for 30 minutes, and it can be observed under a UV light of 254 nm. The position of the orange-red fluorescent band is a DNA band, or is photographed under ultraviolet light. Electropherogram. Ethidium bromide is a carcinogen and should be handled with care. Gloves must be worn. Shanghai Chuangsai Technology provides UV-II portable UV lamp, UV analyzer, commodity number: C71-UV-II-table, the price is 1050 yuan.
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