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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 complex operation, expensive equipment, troublesome gel electrophoresis, etc., and achieve the effect of high application popularity and simple operation

Inactive Publication Date: 2011-08-10
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] Embodiment 1: Color PCR kit qualitative detection effect experiment, determine the minimum value of the detection polymerase chain reaction product, take lambda DNA as the template as an example, carry out the PCR amplification of 500bp target fragment, but not limited to this.

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

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

[0060] 1. Agarose gel electrophoresis detection:

[0061] The amount of PCR product was 100ng / uL, diluted 100 times, the concentration was 1ng / uL, agarose gel electrophoresis (1.0%). 2uL, 1uL, 0.5uL, 0.1uL.

[0062] Electrophoresis results such as Figure 4 shown, by Figure 4 It can...

Embodiment 2

[0066] Example 2. Experiment of quantitative detection effect of color PCR kit

[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, 10x buffer 3uL, T to the EP tube 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 of component A, react at 60℃ for 10 minutes, add 80uL of component B, react at 60℃ for 20 minutes, measure OD 490 , calculate ΔOD=OD 490 -OD 空白 , the results are shown in the table and Image 6 shown.

[0069]

[0070]

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

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PUM

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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] The detection methods of polymerase chain reaction (PCR) products are qualitative and quantitative (including semi-quantitative methods). 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 useful for measuring product accumul [0004] (2) R.Hisada, T.Yagi, 1-Methoxy-5-Methylphenazinium Methyl Sulfate APhotochemically Stable Electron Mediator between NADH and Various Electron Acceptors, J.Biochem., 1977, 82(5), 1469-1473. [0005] (3) Yagi Tatsuhiko, 1-メトキシ PMS, Dojin News, 1979, 14, 1. [0006] (4) S.Nakamura, K.Arimura, K.Ogawa...

Claims

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Application Information

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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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