Determination of the content of nucleic acid by UV absorption method

related topics .Objective
To learn the principle and operating method of determining nucleic acid content by UV phosphorescicity method, and to become familiar with the basic principles and methods of use of UV
remetric photometrics

II, principle
nucleic acids,
nucleotides
and its derivatives in the molecular structure of the radon, the base has a conjugated bi-health system (-C-C one C-C-I), can strongly absorb 250 to 280nm wavelengths 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.
the variation of radon and zirconium base in different pH solutions, 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 oscipation 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 a 260nm wavelength 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, ε (ρ) of 6,000 to 8,000
RNA, 7,000 to 10,000

(ρ) of the calf thymus DNA sodium salt solution ε (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 to 7 800, the RNA has a phosphorus content of 9.5%, and the light density of the RNA solution containing 1 sg /mL RNA is 0.022 to 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. Therefore, the amount of nucleic acids in an unknown concentration of DNA
(RNA) solution can be calculated by measuring the optical density of OD260nm. 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 hydrolysing, which is known as the hyperchromic effect. In
nucleic acids
molecules, hydrogen and π-bonds interact to alter the resonant 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, major instruments and
reagents
1. Test material: NUIC sample DNA or RNA.
2. Main instruments
(1) Analysis
balance
(2) UV tradism meter
(3) ice bath or refrigerator
(4)
centrifuge
(5) centrifuge tube (10mL)
(6)
berrible

<7> (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.
3. Reagent
(1) 5% to 6% ammonia: dilute 5 times with 25% to 30% ammonia.
(2) ammonium niobate-perchloric acid reagents: 3.6mL70% perchloric acid and 0.25g ammonium diphate dissolved in 96.4mL distilled water.
, the operation step
1. The determination of nucleic acid sample purity
(1) accurately called to take the nucleic acid sample to be tested 0.5g, plus a small amount of distilled water (or no ionized water) into a paste, and then add the appropriate amount of water, with 5% to 6% ammonia water to pH7, fixed capacity to 50mL.
(2) take two centrifugal tubes, add 2mL sample solution and 2mL distilled water to the tube, and add 2mL sample solution and 2mL sedimentation agent (precipitation removes large molecular nucleic acids as a control). Mix well and place 30min in an ice bath (or fridge) and 3,000r/min centrifuge 10min. Take 0.5mL of liquid from two tubes, A and B respectively, and use distilled water to determine the capacity to 50mL. A quartz color cup with a light range of 1cm is selected to measure its light density at a wavelength of 260nm.
(3) calculate the .If the nucleic acid sample to be tested does not contain acid-soluble nucleotides or dialysis of low-polynucleotides, the sample can be made into a certain concentration of solution (20 to 50 μg/mL) directly measured on the UV estrphotometer.
2. Determination of nucleic acid solution content
When the nucleic acid sample to be tested contains acid-soluble nucleotides or dialysis of low-polynucleotides, ammonium-perchloric acid precipitation agent is required for determination, precipitation removes large molecular nucleic acids, and the light density at 260 nm of the liquid is determined as a control.
(1) take 2 centrifugal tubes, each tube to add 2mL of nucleic acid solution to be tested, then add 2mL distilled water to the tube, and add 2mL sediment to the tube. Mix well and place 10min in an ice bath (or fridge), 3,000r/min centrifuge 10min. The A and B tubes were diluted to light density between 0.1 and 1.0, respectively. A quartz color cup with a light range of 1cm is selected to measure its light density OD260nm at a wavelength of 260nm.
(2) calculate the .V. Thinking questions
1. What are the advantages and disadvantages of using ultraviolet light absorption to determine the nucleic acid content of the sample?
2. If the sample contains nucleosides
acids
impurities, how should I correct them?
.