分光光度法测定DNA的浓度和纯度(国外英文资料).docVIP

分光光度法测定DNA的浓度和纯度(国外英文资料) Spectrophotometry determines the concentration and purity of DNA 【 objective 】 Understand the principle of measuring the concentration and purity of DNA spectrophotometry; Mastery: the technique of measuring the concentration and purity of DNA by spectrophotometry; Familiar: spectrophotometry for the determination of DNA concentration and purity. 【 experimental principle 】 : In front of the concentration of the extraction of DNA and purity are unknown, in the subsequent DNA enzyme, connection and transformation experiment needs certain concentration and purity requirements, so to measure the concentration and purity of DNA. The methods of measuring DNA usually have: ultraviolet spectrophotometry; AGAR gel electrophoresis (also called spectrophotometry) (1) ultraviolet spectrophotometry: Composition of DNA bases all have certain features for it to absorb uv light, maximum absorption wavelength in between 250 ~ 270 nm, maximum uv absorption value of adenine in 260.5 nm, cytosine: 267 nm, guanine: 276 nm, thymine: 264.5 nm, uracil: 259 nm. These bases will not change the maximum absorption peak of the nucleotides after the nucleotide, but the maximum absorbing wavelength of the nucleic acid is 260nm and the lowest absorption rate is 230nm. This physical property provides the basis for determining the concentration of nucleic acid solution. At the wavelength of 260nm, the optical density of 10OD is equivalent to 50 mu g/ml of double stranded DNA; Single stranded DNA or RNA is 40 mu g/ml; Single stranded oligonucleotide is 20 mu g/ml. You can calculate the concentration of the nucleic acid sample. Spectrophotometry not only determines the concentration of the nucleic acid, but also can determine the purity of the nucleic acid by measuring the ratio of uv absorption of 260nm and 280nm (A260 / A280). The ratio of DNA is 1.8, and the ratio of RNA is 2.0. If the DNA ratio is higher than 1.8, the RNA in the preparation is not yet exhausted. The amoun