Determination of the content of nucleic acid by UV absorption method

I. Objective
To learn the principle and operation method of determining the content of
nucleic acid
by ultraviolet phosphorescicity method, and to become familiar with the basic principles and methods of use of UV phosphorescometers.
II, principle
nucleic acids,
nucleotides
and their derivatives in the molecular structure of radon, steroid base has a conjugated bi-health system (-C-C-C-C-), can strongly absorb 250-280nm wavelength of ultraviolet light. The maximum UV absorption
nucleic acids
DNA, RNA) is at 260nm. In accordance with Lambert-Beer's law, the content of nucleic acids can be determined by changes in the absorption value of ultraviolet light.
different situations in different pH solutions, the variation of radon and zirconium base is different, and the UV absorption light also shows obvious differences, and their molar deconstruction coefficients are also different. Therefore, the determination of nucleic acid substances should be carried out in a fixed pH solution.
the mole-lighting coefficient (or absorption coefficient) of nucleic acids, usually expressed as ε (ρ), i.e. the de-lighting value (i.e. light density, or light absorption) at 260nm wavelengths of a solution containing one mole of phosphorus of nucleic acid per litre. The molar ancticity coefficient of nucleic acids is not a constant, but depends on changes in material preprocessing, pH of the solution, and ion strength. Their classic values (pH s 7.0) are as follows: ε (ρ) of
DNA s 6 000 - 8 000
RNA ε (ρ) s 7 000 - 10 000
ρ ε (ρ) of the calf thymus DNA sodium salt solution (pH s 7.0) is 6,600, the phosphorus content of DNA is 9.2%, and the solution light density of the solution containing 1 sg/mL DNA sodium salt is 0.020. the ε (ρ) of the RNA solution (pH s 7.0) is 7 700-7 800, the RNA contains 9.5% phosphorus, and the light density of the RNA solution containing 1 sg /mL is 0.022-0.024. When the nucleic acid content is determined by ultraviolet estrephosance, it is generally stipulated that at a wavelength of 260nm, the light density of a DNA solution with a concentration of 1μg/mL is 0.020, while the optical density of an RNA solution with a concentration of 1μg/mL is 0.024.
, the optical density OD
260
nm) of an unknown concentration of DNA (RNA) solution can be determined to calculate the amount of nucleic acids in it. The method is simple, fast and highly sensitive, e.g. the nucleic acid content of 3 μg/mL can be measured. For nucleic acid samples containing trace amounts
proteins
and nucleotides and other absorbing ultraviolet light substances, the measurement error is small, if the sample is mixed with a large number of the above-mentioned absorption of ultraviolet light substances, the measurement error is large, should try to remove in advance. The light density of nucleic acids can be increased by about 40% due to degradation or hydrolyzing, which is known as the hyperchromic effect. In the nucleic acids of large molecules, π-bonds interact to alter the resonance behavior of the base. Therefore, the light density of the nucleic acid is lower than the light density of the nucleotides that make up it, a phenomenon known as the hypochromic effect.
, experimental materials, main instruments and
reagents
1. Test material: nucleic acid sample DNA or RNA.
2. Main instrument
(1) Analysis
balance
(2) UV tradisbee photomeometer
(3) ice bath or refrigerator
(4)
centrifuge
(5) centrifuge tube (10mL)
(6)
Cup
(10mL)
(7) capacity bottle (50mL, 100mL)
(8) pipe (0.5ml, 2mL and 5mL)
(9) drug spoon and glass rod
(10) test tube and test tube holder.
.