GB/T 34796-2017 English PDF
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GB/T 34796-2017: Quantification and purity analysis of nucleic acid concentration in solution -- Ultraviolet spectrophotometry
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GB NATIONAL STANDARD OF THE PEOPLE’S REPUBLIC OF CHINA ICS 07.080 A 40 Quantification and purity analysis of nucleic acid concentration in solution - Ultraviolet spectrophotometry Issued on: NOVEMBER 01, 2017 Implemented on: MAY 01, 2018 Issued by. General Administration of Quality Supervision, Inspection and Quarantine; Standardization Administration of the People’s Republic of China.
Table of Contents
Foreword... 3 1 Scope... 4 2 Normative references... 4 3 Abbreviated terms... 4 4 Principle... 4 5 Reagents and materials... 5 6 Main instruments and equipment... 5 7 Sample preparation... 6 8 Detection of nucleic acid concentration and purity by absorbance... 6 9 Detection of nucleic acid concentration and purity by scanning absorption spectroscopy... 8 Appendix A (Informative) Properties of nucleoside triphosphates... 10 Appendix B (Informative) Spectrophotometric results of purified DNA... 11 Appendix C (Informative) Nucleic acid ultraviolet absorption spectrum... 12 Quantification and purity analysis of nucleic acid concentration in solution - Ultraviolet spectrophotometry1 Scope
This Standard specifies the principle, sample preparation, and detection method for detecting the concentration and purity of nucleic acids in aqueous solutions using ultraviolet spectrophotometry. This Standard applies to the detection of nucleic acid concentration and purity in aqueous solutions, as well as to the quantitative determination and purity analysis of nucleic acid samples in aqueous solutions with concentrations of 5 ng/μL and above.2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are indispensable for its application. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. GB/T 6682, Water for analytical laboratory use - Specification and test methods GB/T 11165, pH meter GB/T 26813, Double beam UV/VIS spectrophotometer JJG 646, Verification regulation of locomotive pipette3 Abbreviated terms
For the purposes of this document, the following abbreviated terms apply. DNA. deoxyribonucleic acid RNA. ribonucleic acid4 Principle
Nucleic acids contain conjugated double bonds between purine and pyrimidine bases, which have the property of absorbing ultraviolet light, with the maximum absorption value at 250 nm ~ 270 nm. After the base forms a nucleotide with pentose and phosphate, its maximum absorption peak does not change. The maximum absorption wavelength is about 260 nm, and the absorption low peak is at 230 nm. At a wavelength of 260 nm, the optical density of 1 unit OD value is equivalent to a concentration of 50 μg/mL for double-stranded DNA, 40 μg/mL for single-stranded DNA and RNA, and 33 μg/mL for single-stranded oligonucleotides. This quantity-concentration relationship can be used to calculate the concentration of nucleic acid samples.5 Reagents and materials
Unless otherwise specified, all reagents used in this method shall be analytical reagents, and the water shall be Grade-I water as specified in GB/T 6682. 5.1 DNA standard solution Calf thymus DNA standard solution. 5.2 Tris-hydroxymethylaminomethane buffer (1 mol/L Tris-HCl, pH 7.4) Weigh 121.1 g of Tris into a 1 L beaker; add 800 mL of deionized water; stir thoroughly to dissolve; use NaOH to adjust the pH; bring the volume to 1 L; dispense; autoclave at 103 kPa (1.05 kg/cm2) for 20 min; store at room temperature for later use. 5.3 EDTA (500 mmol/L, pH 8.0) Weigh 186.1 g of Na2EDTA·2H2O; place it in a 1 L beaker; add about 800 mL of deionized water; stir thoroughly; use NaOH to adjust the pH to 8.0 (EDTA can only be completely dissolved at pH 8.0); fix the volume to 1 L; dispense into containers; autoclave at 103 kPa (1.05 kg/cm2) for 20 minutes; store at room temperature for later use. 5.4 TE buffer (10×TE Buffer, pH 7.4) Measure 100 mL of 1 mol/L Tris-HCl (pH 7.4) and 20 mL of 500 mmol/L EDTA (pH 8.0) into a 1 L beaker; add 800 mL of deionized water; mix well; bring the volume to 1 L; after dispensing, autoclave at 103 kPa (1.05 kg/cm2) for 20 min; store at room temperature for later use.6 Main instruments and equipment
6.1 Ultraviolet spectrophotometer or nucleic acid protein analyzer, which shall meet the requirements of GB/T 26813. 6.2 Vortex generator. 6.3 Electronic balance. accuracy, 0.1 mg and 0.01 mg, respectively. 6.4 pH meter, grade 0.1, which shall comply with the requirements of GB/T 11165. 6.5 Adjustable pipettes. 0.5 μL ~ 10 μL, 10 μL ~ 100 μL, 100 μL ~ 1 000 μL, which shall comply with the requirements of JJG 646.7 Sample preparation
Dissolve an appropriate amount of nucleic acid sample in sterile water or TE solution; dilute it to the working curve range for measurement.8 Detection of nucleic acid concentration and purity by
absorbance 8.1 Instrument setup conditions and procedures 8.1.1 Nucleic acid concentration detection Detection wavelength. 260 nm. 8.1.2 Nucleic acid purity detection Detection wavelengths. 260 nm, 230 nm, 280 nm. 8.2 Determination Take an appropriate amount of nucleic acid solution (solution volume depending on the instrument requirements) for detection; use diluted solution to zero; calculate the concentrations of DNA double strand, DNA single strand, and RNA based on the absorbance of nucleic acid at wavelengths of 260 nm, 230 nm, and 280 nm; determine the purity of nucleic acid based on the OD260/OD280 and OD260/OD230 ratios. Repeat the measurement three times and take the average value. The properties of nucleoside triphosphates are shown in Table A.1, and the spectrophotometric results of purified DNA are shown in Table B.1. 8.3 Result calculation 8.3.1 Calculation of nucleic acid concentration detection results Calculate nucleic acid concentration using the following formula. a) The molar concentration of nucleic acid samples is calculated according to Formula (1). ......Source: Above contents are excerpted from the full-copy PDF -- translated/reviewed by: www.ChineseStandard.net / Wayne Zheng et al.
