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PCR (Polymerase Chain Reaction) product qualitative and semiquantitative detecting kit

A semi-quantitative detection and kit technology, applied in the biological field, can solve the problems of complicated operation, expensive instruments, time-consuming and labor-intensive, etc., and achieve the effect of high application popularity and simple operation.

Inactive Publication Date: 2012-12-12
内蒙古龙鑫生物科技有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for the detection of a large number of samples, gel electrophoresis is cumbersome, time-consuming, labor-intensive and expensive
Quantitative detection by real-time fluorescent quantitative PCR method requires expensive instruments, complex operation, and limited by site conditions, and poor application popularity

Method used

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  • PCR (Polymerase Chain Reaction) product qualitative and semiquantitative detecting kit
  • PCR (Polymerase Chain Reaction) product qualitative and semiquantitative detecting kit
  • PCR (Polymerase Chain Reaction) product qualitative and semiquantitative detecting kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1: Qualitative detection effect experiment of color PCR kit to determine the minimum value for detection of polymerase chain reaction products Taking lambda DNA as a template as an example, PCR amplification of a 500bp target fragment was performed, but it is not limited thereto.

[0058] PCR system: 10μl of 10×PCR buffer, 15mM MgCl 2 4 μl, 8 μl of 2.5mM dNTPmix, 1 μl each of 20 μM lambda1primer and lambda 2primer, 20-200 pg of template DNA, 0.5 μl of Taq DNA polymerase (5U / μl), and replenish water to 100 μl.

[0059] The reaction conditions are: pre-denaturation at 94°C for 30 seconds; denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, elongation at 72°C for 30 seconds; 25 cycles.

[0060] 1. Agarose gel electrophoresis detection:

[0061] The amount of PCR product is 100ng / uL, diluted 100 times, the concentration is 1ng / uL, agarose gel electrophoresis (1.0%), the sample volume is respectively: 100uL, 50uL, 20uL, 15uL, 10uL, 8uL, 6uL, 4uL, ...

Embodiment 2

[0066] Embodiment 2, color PCR kit quantitative detection effect experiment

[0067] 1. Take the quantitative detection of T4 DNA ligase (Ligase) activity (three batches: K2900, K3000, K3100) as an example to confirm the quantitative detection effect of the color PCR kit.

[0068] Add 5nM pBgl II Linker 15uL, 10 times buffer 3uL, T 4 DNA ligase (different concentrations) 1uL, supplemented with water to 30uL, reacted at 16°C for 70 minutes, heated at 90°C for 5 minutes, and added 70uL of water. Then add 20uL component A, react at 60°C for 10 minutes, add 80uL component B, react at 60°C for 20 minutes, measure OD 490 , calculate ΔOD=OD 490 -OD 空白 , the results are shown in the following table and Image 6 shown.

[0069]

[0070]

[0071] The test results of the three batches were 353U / μl for K2900, 345U / μl for K3000, and 358U / μl for K3100. The results of the three batches were very close, indicating that the color PCR kit can be used for the quantitative determinatio...

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Abstract

The invention relates to the technical field of biology, in particular to a PCR (Polymerase Chain Reaction) product qualitative and semiquantitative detecting kit (color PCR kit). The kit comprises a plurality of reagents, such as inosine monophosphate, xanthine oxidase, iodine chloride nitro-tetrazole, hypoxanthineguanine phosphoribesyltransferase, pyrophosphoric acid, and the like. The invention can be used for qualitatively or semiquantitatively detecting a PCR product.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a PCR product qualitative and semi-quantitative detection kit (color PCR kit). Background technique [0002] There are two methods for detection of polymerase chain reaction (PCR) products: qualitative and quantitative (including semi-quantitative). method to complete the determination of PCR products. Both methods measure the nucleic acid portion of the polymerase chain reaction (PCR) product. The current technology can refer to the following documents: [0003] (1) Tagiri-Endo M, A colorimetric assay for inorganic pyrophosphate that is also sufficient for measuring product accumulation in polymerase chain reactions. Anal. Biochem., 2003, 315(2): 170-4. [0004] (2) R. Hisada, T. Yagi, 1-Methoxy-5-Methylphenazinium Methyl Sulfate A Photochemically Stable Electron Mediator between NADH and Various Electron Acceptors, J. Biochem., 1977, 82(5), 1469-1473. [0005] (3) Tatsuhiko Yag...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 陈永胜翟景波李国瑞
Owner 内蒙古龙鑫生物科技有限责任公司
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