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Quantitative plasmid DNA (Deoxyribonucleic Acid) detection kit

A quantitative detection and kit technology, applied in the field of biochemical analysis, can solve problems such as lack of uncertainty information and traceability, lack of plasmid DNA, failure to meet requirements, etc., and achieve high accuracy, high standard accuracy, and uncertain low degree of effect

Inactive Publication Date: 2013-03-20
NAT INST OF METROLOGY CHINA
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the limitations of the PicoGreen fluorescent dye method in the quantification of DNA are as follows: first, the accuracy of the method depends on the accuracy of the DNA standard content in the kit, and the DNA standard in the currently commercially available kits The content value is a reference value provided by the manufacturer, that is? ng / ml, this value is generally determined by ultraviolet absorption (OD260), and there is no relevant uncertainty information and traceability for this value
Second, because PicoGreen has different DNA binding efficiencies of different molecular sizes, if there is a large difference between the quantitative DNA standard and the measured DNA sample molecular size, it will lead to a large deviation in the quantitative results of the sample. Currently, commercial There is only one molecular size DNA standard in all kits, that is, Lambda phage DNA standard (48.5K)
Therefore, if the plasmid DNA is quantified by the PicoGreen method, the existing commercial kits cannot meet the requirements due to the lack of plasmid DNA standards
[0004] Because the quantification of plasmid DNA by the PicoGreen fluorescent dye method requires plasmid DNA standard substances with accurate quantities, and currently there is no plasmid DNA standard with accurate quantities that can be used as a standard for the quantification of plasmids by the PicoGreen method

Method used

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  • Quantitative plasmid DNA (Deoxyribonucleic Acid) detection kit
  • Quantitative plasmid DNA (Deoxyribonucleic Acid) detection kit
  • Quantitative plasmid DNA (Deoxyribonucleic Acid) detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] The relationship between embodiment 1 plasmid DNA and PicoGreen fluorescence signal

[0076] 1. Purpose of the experiment:

[0077] To explore whether there is a linear relationship between the concentration of plasmid DNA and the fluorescence signal of PicoGreen

[0078] 2. Materials and methods:

[0079] 1. Plasmid DNA pBAZS: obtained by ligating four exogenous gene fragments on the vector pEASY-T3 (see Table 1 for the primers used for amplification).

[0080] 2. PicoGreen fluorescent dye: purchased from life technology company. Dilute 1:200 with 1xTE.

[0081] 3. Prepare the plasmid DNA to a storage concentration of 10000ng / mL, then dilute to 1000ng / mL, 750ng / mL, 500ng / mL, 250ng / mL, 100ng / mL, 10ng / mL, 1ng / mL, 0.5ng / mL with 1xTE mL, 0.25ng / mL.

[0082] 4. Mix 0.1mL of the diluted DNA solution with 0.1mL of the diluted PicoGreen dye, and transfer to a 96-well microtiter plate.

[0083] 5. Place the microplate plate on the microplate reader, select the excitation ...

Embodiment 2

[0090] Embodiment 2PicoGreen method is to the determination of the linear range of plasmid DNA quantification and limit of quantification and detection sensitivity

[0091] 1. Materials and methods

[0092] 1. Prepare the plasmid DNA at a storage concentration of 10000ng / mL, then dilute to 5000ng / mL, 2500ng / mL, 2000ng / mL, 1000ng / mL, 750ng / mL, 500ng / mL, 250ng / mL, 100ng / mL with 1xTE , 10ng / mL, 1ng / mL, 0.5ng / mL, 0.25ng / mL, 0.05ng / mL, 0.01ng / mL.

[0093] 2. Mix 0.1mL of the diluted DNA solution with 0.1mL of the diluted PicoGreen dye, and transfer to a 96-well microtiter plate. Other operations are the same as 5 and 6 in the implementation example 1.

[0094] 2. Experimental results:

[0095] 1. Determination of the linear range of plasmid DNA by PicoGreen fluorescent dye method

[0096] In order to determine the linear range of plasmid quantification by the PicoGreen fluorescent dye method, DNA was diluted in 14 different concentration gradients, and the relationship between ...

Embodiment 3

[0101] Example 3 Quantification of DNA with different molecular sizes using the PicoGreen fluorescent dye method

[0102] 1. Purpose of the experiment:

[0103] To study whether the commercially available detection reagent Lambda DNA can be used as a standard for the determination of plasmid DNA by fluorescent dye method.

[0104] 2. Materials and methods:

[0105] 1. Select DNA of different molecular sizes: lambda genomic DNA, size 48502bp, purchased from TAKARA company, 9000bp DNA: use EcoRI restriction endonuclease to digest Lambda genomic DNA, recover and purify by agarose coagulation electrophoresis; pBAZS plasmid DNA, as above mentioned. nosZPCR product: using the constructed plasmid as a template and amplifying with primer nosZ-F / R, see Table 1 for specific primer sequences.

[0106] Table 1 Primer Sequence

[0107]

[0108] 2. PicoGreen fluorescent dye: purchased from life technology company. Dilute 1:200 with 1xTE.

[0109] 3. Prepare the DNA of four diffe...

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Abstract

The invention provides a quantitative plasmid DNA (Deoxyribonucleic Acid) detection kit which has the characteristics of adopting a plasmid DNA standard substance to accurately quantify by using an ultrasonic-isotope dilution mass-spectrography with high accuracy, and making up the defect that a conventional kit cannot quantify the plasmid DNA. The invention further provides a plasmid DNA quantitative detection method which comprises the following steps of: respectively measuring fluorescence signal strengths of the plasmid DNA standard substances of different concentrations; drawing a concentration-fluorescence signal strength standard curve; subsequently measuring the fluorescence signal strength of a plasmid DNA sample to be measured; and accurately calculating the DNA concentration of the sample to be measured according to the drawn standard curve. According to the method and the kit, standard plasmids are adopted to be used as the DNA standard to quantify in a fluorescent dye method for the first time with high sensitivity, and DNA as low as 1pg can be detected; the linear range is wide, and plasmid DNA of 0.25-1000ng / Ml can be detected; and the standard plasmids are quantified by using the ultrasonic-isotope dilution mass-spectrography, and the quantitative value obtained is low in uncertainty and high in accuracy.

Description

Technical field: [0001] The invention belongs to the field of biochemical analysis, and in particular relates to a plasmid DNA fluorescent dye quantitative detection method and a detection kit. Background technique: [0002] Plasmid DNA is a small (1-200kb) covalent, closed, circular double-stranded DNA molecule free from extrachromosomal, which can autonomously replicate and stably inherit genetic factors. It usually encodes some enzyme genes, such as antibiotics, antibiotic resistance, restriction enzymes, modification enzymes and other enzyme genes that are beneficial to the host. Because of its small size, autonomous replication and stable inheritance, it is a commonly used carrier in molecular biology and genetic engineering technology. DNA analysis in the field of molecular biology often requires accurate quantification of plasmid DNA. The concentration of plasmid DNA was accurately determined. Therefore, the quantification of plasmid DNA has a wide range of applica...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
Inventor 董莲华孟盈王晶傅博强高运华张玲
Owner NAT INST OF METROLOGY CHINA
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