DNA quantitative detection method and kit using SYBR Green fluorescent dye

A quantitative detection method and fluorescent dye technology are applied in the field of SYBR Green I fluorescent dye DNA quantitative detection kits to achieve the effect of wide linear range, low uncertainty and high sensitivity

Inactive Publication Date: 2014-06-18
王泽广
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The development of SYBRGreen I quantification kits for DNA requires DNA standard substances with accurate values. At present, there is no DNA standard with accurate values ​​that can be used as a standard for the quantification of plasmids by the SYBR Green I method.

Method used

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  • DNA quantitative detection method and kit using SYBR Green fluorescent dye
  • DNA quantitative detection method and kit using SYBR Green fluorescent dye
  • DNA quantitative detection method and kit using SYBR Green fluorescent dye

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1 Quantification of DNA with different molecular sizes using the SYBR Green I fluorescent dye method

[0060] 1. Materials and methods:

[0061] 1. DNA of different molecular sizes: lambda genomic DNA, 48kb in size, purchased from Takara Company; 9kb DNA: digested with EcoRI restriction endonuclease, and recovered and purified by agarose coagulation electrophoresis; plasmid DNA: 5kb ; PCR product: 200bp.

[0062] 2. SYBR Green I fluorescent dye was diluted 1:10000 with 1xTE.

[0063] 3. Prepare the DNA of four different molecular sizes mentioned in 1 to a storage concentration of 10000ng / mL, and then dilute to 2000ng / mL, 1000ng / mL, 500ng / mL, 200ng / mL, 20ng / mL, 2ng with 1xTE / mL, 0.5ng / mL.

[0064] 4. Mix 0.1mL of the diluted DNA solution with 0.1mL of the diluted SYBR Green I dye, and transfer to a 96-well microtiter plate.

[0065] 5. Put the microplate plate on the microplate reader, select the excitation wavelength of 480nm and the emission wavelength of ...

Embodiment 2

[0077] Embodiment 2 SYBR Green I method is to the determination of the linear range and limit of quantification and detection sensitivity of DNA quantification

[0078] 1. Materials and methods

[0079] 1. Prepare the plasmid or genomic DNA to a storage concentration of 10000ng / mL, then dilute to 5000ng / mL, 2500ng / mL, 2000ng / mL, 1000ng / mL, 750ng / mL, 500ng / mL, 250ng / mL, 100ng with 1xTE / mL, 10ng / mL, 1ng / mL, 0.5ng / mL, 0.25ng / mL, 0.05ng / mL, 0.01ng / mL, 0.005ng / mL.

[0080] 2. Mix 0.1mL of the diluted DNA solution with 0.1mL of the diluted SYBR Green I dye, and transfer to the same 96-well microtiter plate. Other operations are the same as 5 and 6 in the implementation example 1.

[0081] 2. Experimental results:

[0082] 1. Determination of the linear range of plasmid DNA by SYBR Green I fluorescent dye method

[0083] In order to determine the linear range of plasmid quantification by the SYBR Green I fluorescent dye method, the DNA was diluted in 15 different concentration g...

Embodiment 3

[0094] Example 3 Digital PCR method quantifies the standard

[0095] 1. Template Preparation

[0096] Measure the concentration of 5k plasmid DNA and lambda genomic DNA by ultraviolet method, convert the obtained concentration from ng / μL to copy / μL according to the formula (1), and then weigh the DNA through a balance with TE 0.1 Dilute to 1200copies / μl for later use.

[0097] w = C × 6.02 × 10 23 B × 660 - - - ( 1 )

[0098] w: DNA concentration to be tested, copy / μL;

[0099] C: the DNA concentration measured by ultraviolet method;

[0100] B: The number of DNA bases to be tested.

[0101] 2. dPCR analysis

[0102]1) The dPCR reaction system for plasmid DNA and genomic DNA is co...

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Abstract

The invention relates to a DNA quantitative detection method and a detection kit. The DNA quantitative detection method establishes the conditions of SYBR Green I fluorescent dye method for DNA quantification to obtain the linear range and sensitivity of the method, and the accurate content of a DNA sample can be fast and accurately obtained. According to the DNA quantitative detection method, plasmid molecules and genomic DNA standard substances are also constructed and prepared, two DNA standard substances are accurately quantified by use of a digital PCR (polymerase chain reaction) method, the standard plasmid and genomic DNA can be used for gradient dilution to obtain a standard curve of the SYBR Green I fluorescent dye method for the DNA quantification, and the accurate content of a to-be-tested DNA sample can be obtained according to the standard curve.

Description

Technical field: [0001] The invention relates to a method and a detection kit for detecting genomic DNA and plasmid DNA, in particular to a SYBR Green I fluorescent dye DNA quantitative detection kit. Background technique: [0002] Accurate quantification of genomic and plasmid DNA is often required for DNA analysis in the field of molecular biology. [0003] Nucleic acid quantification using PicoGreen fluorescent dye method is widely used because of its high sensitivity and wide linear range. However, the PicoGreen fluorescent dye is expensive, and its limitation in quantifying DNA is that the accuracy of the method depends on the accuracy of the DNA standard content in the kit, while the DNA standard content in the currently commercially available kits The content value is a reference value provided by the manufacturer. This value is generally determined by ultraviolet absorption (OD260), and there is no relevant uncertainty information and traceability. Secondly, becaus...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6851G01N21/6428C12Q2531/113C12Q2563/107C12Q2545/114
Inventor 王泽广吴立娟董杰
Owner 王泽广
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