Ultraviolet phosphorescies determine the content of nucleic acids

, objective,
1, to understand the basic principles and methods of use of UV ionolight meters.
2, learn the principle and operation method of determining the
content of
nucleic acids by ultraviolet phosphorescies.
II, principle
nucleic acids,
nucleotides
and their derivatives in the molecular structure of radon, steroid base has conjugated bi-health system (-C-C one C-C-I), can strongly absorb 250 to 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. In different pH solutions, the variation of radon and nirconium 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-light-absorbing coefficient (or absorption coefficient) of nucleic acids, usually expressed as the anti-light value (i.e., light density, or photosorption) at a wavelength of 260nm per litre of solution containing one mole of phosphorus of nucleic acid. The molar ancticity coefficient of nucleic acids is not a constant, but depends on changes in the pre-treatment of the material, the pH of the solution, and the ion strength. Their classic values (pH s 7.0) are as follows:
DNA of the s epsilon
; The light density of the solution containing 1
/mL RNA is 0.022 to 0.024. When the nucleic acid content is measured by ultraviolet estrephosance, it is usually stipulated that at a wavelength of 260nm, the optical density of a DNA solution with a concentration of 1 sm;g/mL is 0.020, while the optical density of an RNA solution with a concentration of 1 sm;g/mL is 0.024. Therefore, the optical density OD260nm of the unknown concentration of DNA (RNA) solution can be determined to calculate the amount of nucleic acids in it.
simple, fast and high sensitivity, such as nucleic acid content of 3 sm;g/mL can be measured. For nucleic acid samples containing trace amounts of
proteins
and nucleotides, such as UV-absorbing 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.
3, equipment
1,
analysis
balance
2,
; 3,
capacity bottle
;4, ultraviolet phosphorescometer; 5, straw
6, ice bath or refrigerator
four,
reagents1, 5% to 6% ammonia
2, ammonium diphnate - chloric acid reagents (precipitation agent). If the preparation of 200mL can be added to 193mL distilled water
7mL70% perchloric acid and 0.5g ammonium tantalum.
, operation
1, with the analysis balance accurately called to take the nucleic acid sample to be tested 500 mg, plus a small amount of distilled water into a paste, and then add a small amount of water dilution. Then with 5% to 6% ammonia to pH7, fixed capacity to 50mL.
2, take 2 centrifugal tubes, add 2mL sample solution and 2mL distilled water to the first tube, and add 2mL sample solution and 2mL sediment to the second tube to remove the large molecular nucleic acid as a control. Mix well. Centrifuge (3000r/min, 10 minutes) after 30 minutes in an ice bath or refrigerator. From the first tube and the second tube, respectively, absorb 0.5mL of liquid, with distilled water to 50mL. A quartz color cup with a light range of 1cm measures its light absorption value (A1 and A2) at a wavelength of 260nm.
6. Calculation
If the nucleic acid sample to be tested is known to contain no acid-soluble nucleotides or dialysis of low-polynucleotides, the sample can be disprosided into a certain concentration of solution (20 to 50 kg/mL) directly measured on the UV hydrophosmeter.