Experimental application scheme of DNA nucleic acid concentration determination （Microscopic UV spec

Introduced oligonucleotides, single-stranded, double-stranded DNA, as well as RNA can be quantitatively dissolved in the buffer，The purine and pyrimidine bases that constitute nucleic acids have conjugate double bonds,Make the nucleoside nuclei and nucleic acids in ultraviolet light with a characteristic absorption spectrum,The maximum absorption peak was 260nm,Narrow-light-band UV-spectrophotometer,Colorimetric cup light diameter is 1cm,One absorbance value (1A) is equivalent to 50 u g/ml double-screw DNA,40 u g/ml single-helical DNA or RNA),The 20 u g/ml oligonucleotide .

Principles of UV spectrophotometry based on the benzene ring structure of the bases on the DNA chain has a strong absorption in the purple light region，The DNA / RNA has the maximum absorption peak at 260nm,The protein has the maximum absorption peak at 280nm,Salts and small molecules were concentrated at 230nm.Therefore, the nucleic acid concentration can be measured by spectroscopy at a 260nm wavelength,An OD of 1 corresponds to approximately 50 u g/ml of duplex DNA,The OD value of 1 corresponds to approximately 50 u g/ml of double-stranded DNA, with a single-stranded DNA concentration of about 33ug l ml, an RNA of about 40 u g/ml, and an oligonucleotide of about 35ug / ml.If using a 1cm light diameter,DNA / RNA samples were diluted n-fold with H, O and with H, O as blank control,The concentration before dilution was calculated from the OD260 value read out at this time.

DNA (mg/ml) =50*OD260 reading*dilution / 1000RNA (mg/ml) =40*OD260 reading*dilution / 1000.

A280nm is the absorption wavelength of the highest absorption peak of protein and phenols, and the ratio can be evaluated for the purity of the nucleic acid sample: the A260 / A280 ratio of pure DNA is 1.8, and the pure RNA is 2.0. If the ratio is low, the protein (aromatic)Or contamination of classified substances, requiring purification of the sample. Ratio =1.5 is equivalent to a 50% protein / DNA solution.

A230nm is the absorption wavelength of the highest absorption peak of carbohydrate, and the ratio can be assessed for nucleic acid sample purity: the A260 / A230 ratio of pure DNA and RNA is 2.5.If the ratio is less than 2.0 and indicates that the sample is contaminated with carbohydrates (sugars), salt, or organic solvents, the sample should be purified.

Either A320nm or A340nm is the turbidity of the test solution sample, and the value should be close to 0.0. If insufficient, indicate the suspension in the solution, need to purify the sample.

Instruments and reagents

UL-1000 Ultra UV visible spectrophotometer

cuvette

DNA

DDW

Determine

1. The prepared DNA was suspended in pH8.0,10mmo1L of Tris buffer in TE buffer, containing (1 mmol/L EDTA) or suspended in sterilized water (add water according to DNA content).

2. Determine the UV absorption curve of DNA / RNA by scable UV spectrophotometer, determine OD260 and OD280, and calculate the ratio: OD260 / OD280, the ratio of pure DNA is about 1.8~2.0. This ratio for pure RNA is about greater than 2.0.

3. The purity of the resulting DNA was calculated according to the OD260 value of 1 micrograms of pure DNA =0.020: the OD260 value of 1 micrograms of pure RNA per ml was =0.025, and the purity of the resulting RNA was calculated.

Matters needing attention

The OD values should range between 0.1 and 0.99, otherwise not in the above linear relationship.

The A260 / A280 ratio provides a reference for DNA purity, but the A260 / A280 ratio is affected by the pH. If pH is not adjusted, the ratio may differ greatly from the reality.If accurate values are required, it is recommended to test in 10 mM Tris Cl, pH 8.5, where the pure DNA A260 / A280 ratio should be 1.8-2.0 (note that the same buffer should be used as a control)

During testing, too high ion concentrations can also cause reading drift,Therefore, it is recommended to use a buffer with certain pH value and low ion concentration,Such as TE, it can be greatly stabilized for reading.The dilution concentration of the sample is also an important factor:Because some small particles in the sample, especially nucleic acid samples.The presence of these small particles interferes with the test effect.To minimize the effect of the particles on the test results, the nucleic acid absorbance value is required to be at least greater than 0.1A, preferably at 0.1-1.5A. In this range, the interference of the particles is relatively small, and the results are stable.This means that the concentration of the sample cannot be too low, or too high (beyond the test range of the luminometer).Finally, the operation factors, such as mixing to be sufficient, otherwise the light absorption value is too low, or even a negative value;Mimixture can not exist bubbles, blank liquid no suspended matter, otherwise the reading drift violently;The blank liquid and sample must be used to test with the same colorimetric cup, otherwise the concentration difference is too large;The conversion coefficient and the sample concentration unit selection are consistent;The window wear colorimetric cup cannot be used; the volume of the sample must reach the minimum volume required by the colorimetric cup.

 Instrument parameter

UL-1000 Ultra UV visible spectrophotometer product parameters

1. Product profile

Spectrophotometer is an important analysis instrument wildly applied in research of physics, chemistry, biology, medicine, material, enviroment and modern production management of chemistry, medicine, enviroment test, metallurgy. Spectrophotometer is the instrument for quantitative and quantitative analysis by spectrophotometry. It is usually used in quatitative research of nucleic acid, proteins and bacterial concentration.

Feature

●Microscale sample test, minium 0.5ul

●Wildly test area, 100 times of the traditional spectrophotometer

●No need to dilute for most samples

●Test directly, no need to warm-up, container test and daily consumtion test

●Whole wavelength 190-1100nm, accuracy 1nm, auto scannig wavelength 190-850nm

●Compact size and partable package fitable for on-site test

●More accurate and fexiable test is realized by PC contry

Optical Specifications

About Us

Introduction of Shanghai Macylab Instrument Company

Shanghai Macylab Instrument Co., Ltd (hereinafter referred to as Macylab),Is a high-tech enterprise with independent intellectual property rights,Macylab of the entrepreneurial ideas "Technology —— is changed because of you." And with this as the enterprise purpose, constantly explore, bold innovation.Especially in the field of analysis and testing instruments, advanced products are constantly developed, so that American analysis becomes a supplier of high-quality instrument resources.

Macylab of the main spectroscopic instruments visible spectrophotometer, ultraviolet visible spectrophotometer, atomic absorption spectrometer, ultra-micron spectrophotometer, atomic fluoruminometer, ICP inductively coupled plasma emission spectrometer, ICP inductively coupled plasma mass spectrometer,At present, our products have been widely used in organic chemistry, inorganic chemistry, biochemistry, medicine, environmental protection, metallurgy, petroleum, agriculture and other fields.At the same time, using the rich experience accumulated in product mechanical structure, optical design, electrical application and software development,Combined with the latest actual demand of the market, a batch of new analytical instruments will be launched in the near future.

Our headquarters and production base are located in Shanghai, The marketing center is located in Beijing, and it has built research and development bases in Jiangsu, Shanghai and Shandong. To make full use of the intellectual resources of various regions ,Macylab has also carried out deep scientific research cooperation with some scientific research institutions at home and abroad, constantly transforming scientific research results into productive forces.To better serve the majority of customers,There are 12 offices in China,Customize the application solutions that meet your needs,Increase the added value of products.While constantly serving domestic users, Distribution agencies in more than 20 countries have established deep strategic partnerships.

Macylab instrument is not just a high-tech certification enterprise,More passed the CE certification, FCC certification, RoHS certification and a number of domestic qualification review certification,And has a number of self-developed spectrum patent copyright and so on.