Determination of nucleic acid content - ultraviolet absorption.

determination of nucleic acid
content - ultraviolet absorption methodnucleotides,
nucleotides
, nucleic acids in the composition of the composition of radon, niacin, niacin Base, these bases have conjugated double bonds (-C-C-C-C-C-), in the ultraviolet light region of 250-280nm has a strong light absorption, the maximum absorption value of about 260nm. The UV absorption of nucleic acids is often used for quantitative determination of nucleic acids.(P) of the nucleic ε acid is expressed as the light absorption value of 1 mole of atomic phosphorus per liter of solution. The ε(P) 260nm (pH7.0) of RNA is 7,700 to 7,800, and the RNA contains about 9.5% phosphorus, so the light absorption value of 1 μg RNA per milliliter solution is equivalent to 0.022 to 0.024. The calf thymus
DNA
sodium salt ε(P) 260nm (pH7.0) is 6,600 and has a phosphorus content of 9.2%, so the light absorption value of 1 μg DNA sodium salt per milliliter solution is equivalent to 0.020.the light absorption value at 260nm, the content of nucleic acid can be calculated. When nucleic acid degradation, its UV absorption intensity increases significantly, called the coloration effect.Protein
also has UV absorption, usually the protein absorption peak at 280nm wavelength, at 260nm absorption value is only 1/10 or less nucleic acid, so for nucleic acid samples containing trace amounts of protein, the measurement error is small. If a large number of impurities with ultraviolet absorption are mixed in the nucleic acid products to be tested, the measurement error is large and should be removed. Innocent samples cannot be quantified with UV absorption values.the purity of the sample can be determined by the ratio of A260/A280. Pure RNA A260/A280≥2.0; DNA A260/A280≥1.8. When the protein content in the sample is high, the ratio decreases. The ratio of RNA to DNA is less than 2.0 and 1.8, respectively, indicating that the sample is not pure.pH has an effect on the UV absorption of nucleic acids, so the pH of the solution should be fixed at the time of determination.the nucleic acid content is determined by using the commonly used ratio of light-dissipation coefficient.. Methods and steps are . 1, the determination ofto take clean
centrifugal tube
A B two, respectively, accurately add 1.0mL DNA / RNA sample solution, and then add 1.0mL distilled water to the A tube, to the acetylene to add 1.0 ml of ammonium perchlorate-dichloric acid precipitation agent, shake the rear refrigerator 30min, so that the precipitation is complete. 3000r/min centrifugation 10min, each absorbed liquid 0.5mL into the corresponding A-B two
capacity bottle
, fixed capacity to 50mL. The above-mentioned two diluted A260 values of A-B were measured by distilled water for blank control and ultraviolet photometrics were used. 2, calculating the total DNA/RNA content in thetest fluid press-down calculation:medium A260 - the total light density value of the diluted liquid under test at 260nm; ethyl A260 - add sediment to remove large molecular nucleic acids The light density value of the dilution was measured at 260nm, the difference between the(A260-BA260) was the light density value of the dilution under test, and the VB - the total volume of the tested fluid (mL) D - the dilution multiply of the sample.0.020 - The ratio of DNA to the decontabation coefficient, i.e. the aqueous solution (pH neutral) containing 1 μg of DNA sodium salt per milliliter at a wavelength of 260nm, passing the light density value at 1cm.0.022 - The ratio of ICTs to the light-dissipation coefficient is the light density value at 260nm wavelengths of 1 mg/L ICT aqueous solution (pH neutral) with a light diameter of 1cm.because large molecule nucleic acids are susceptible to degeneration, this value also varies depending on the degree of degeneration, so it is generally used to calculate the amount of DNA or RNA compared to the ocance coefficient is an approximation..