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

Time:2022-11-14  Read:

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 .

Before the test,Select the correct program,The volume of the input stock and the diluent,After the test of the blank liquid and the sample liquid. Yet, The experiment was not plain sailing.Reading instability may be the biggest headache for the experimenter.The higher the sensitivity of the instrument, the greater the light absorption value drift shows.

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





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.


●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

Minimum Sample Size


Wavelength Range


Optical Distance


Wavelength Accuracy


Wavelength Resolution

≤0.3nm(FWHM at Hg 253.7nm)

Absorbance Accuracy

2%(0.76 at 257nm)

Absorbance Resolution

0.002Abs(at 1mm optical distance)

Absorbance Range

0.02-300A10mm  equivalent)

Detection Range

Lower limit 2ng/ul (dsDNA);Upper limit 15000ng/ul (dsDNA)

Sample Pedestal Material

304 Stainless Steal and Quartz

Measurement Time




Light Source

Xenon flash lamp

Absorbance Test Range

0.1~5Abs. ±0.1Abs  5~80Abs.±2% can extend to 300Abs)

Dimensions (W x D x H)mm




Operating Power




Display Language

English and Chinese

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