Chemical determination of DNA content can be used for (1) estimation of plasmid concentration prior to transformation and transfection, and (2) assay of acquired DNA content.

Operation method

Determination of DNA by chemical method - Diphenylamine colorimetric method

Principle

When DNA is heated under acidic conditions, the glycosidic bond between its purine base and deoxyribose is broken to produce purine base, deoxyribose and deoxypyrimidine nucleotides, while 2-deoxyribose is dehydrated by heating in an acidic environment to produce ω-hydroxy-γ-ketopentanose, which reacts with diphenylamine to produce a blue color substance with a maximal absorption at 595 nm.The light absorption of DNA is directly proportional to the concentration of DNA in the range of 40-400 μg. The light absorption of DNA in the range of 40-400 μg is proportional to the concentration of DNA. The sensitivity of the reaction can be improved by adding a small amount of acetaldehyde to the reaction solution.

Materials and Instruments

DNA samples
DNA Sodium salt NaOH solution Diphenylamine Ice acetic acid Perchloric acid Acetaldehyde
Spectrophotometer Water bath

Move

1. Make DNA standard curve: take 12 clean and dry test tubes and add DNA standard solution, after adding each reagent, mix well. Keep warm in 60℃ water bath for 1h, after cooling, zero at 595nm with 1,2 tubes as blank, and determine the optical density of each tube (OD595nm). Take the average value of the two tubes, take the DNA content as the horizontal coordinate and the optical density as the vertical coordinate, and plot the standard curve.

2. Determination of DNA content in sample: Take 2 clean test tubes and add sample reagent, other operations as above. The DNA content in the solution to be measured should be adjusted to within the readable range of the standard curve. 3.

3. Calculation of DNA content: Using the optical density of the sample, find out the corresponding DNA content from the standard curve and calculate the percentage of DNA in the sample.

Caveat

Diphenylamine reagent is corrosive, and the blue color produced by diphenylamine reaction is not easy to fade, operation should be prevented from spilling, colorimetry, the outside of the colorimetric cup must be wiped clean.

Common Problems

Diphenylamine is mainly used as antioxidant for synthetic rubber, fuel and pharmaceutical intermediates, antioxidant for lubricating oil, and stabilizer for gunpowder. Determination of nitrates, chlorates and other oxidizing substances. Catalytic determination of iridium. Photometric determination of nitrates. Redox indicator. Haploid breeding medium. Manufacture of dyes. Stabilizer for nitrocellulose, explosives, fire cotton.

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