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Bovine gapdh gene transcription level fluorescent quantitative PCR detection kit

A detection kit and gene transcription technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems affecting the accuracy of quantification, and achieve the effects of good stability, high sensitivity and low experimental cost

Inactive Publication Date: 2016-01-20
LANZHOU INST OF ANIMAL SCI & VETERINARY PHARMA OF CAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since SYRBGreen binds to all double-stranded DNA, false positives caused by primer-dimers, single-stranded secondary structures, and erroneous amplification products can affect the accuracy of quantification

Method used

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  • Bovine gapdh gene transcription level fluorescent quantitative PCR detection kit
  • Bovine gapdh gene transcription level fluorescent quantitative PCR detection kit
  • Bovine gapdh gene transcription level fluorescent quantitative PCR detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] 1) Prepare the GAPDH fluorescent quantitative PCR reaction solution in proportion, and the 20 μL reaction system is as follows:

[0037] Table 2

[0038]

[0039] Negative control and positive control should be set in the same quantitative reaction.

[0040] 2) Take 1000 μL of 2×SYBRGREENMIX, 100 μL of primer mixture and 800 μL of ultrapure water and mix thoroughly to prepare 1900 μL of FQ-PCR reaction master mix.

[0041] 3) Thoroughly mix the above liquids, divide the premixed liquid into 100 small tubes according to 19 μL / tube, and add them to the special PCR reaction tubes or reaction plates for fluorescence quantification.

[0042] 4) Prepare 5 tubes of standard GAPDH gene template with serial dilutions of 10E+8 / ML, 10E+7 / ML, 10E+6 / ML, 10E+5 / ML, 10E+4 / ML, 10 μL in each tube.

[0043] 5) FQ-PCR amplification Add 1 μL of the cDNA to be tested, water or standard GAPDH gene template to each reaction tube containing 19 μL of the PCR reaction master mix, shake and m...

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PUM

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Abstract

The invention discloses a fluorogenic quantitative polymerase chain reaction (PCR) detection kit for bovine reduced glyceraldehyde-phosphate dehydrogenase (GAPDH) gene transcription levels. The kit comprises the following components: 2*SYBR Green MIX, a primer mixing liquid consisting of primers 1 and 2 which are both 8 micro mol / L, a standard GAPDH gene template and ultrapure water with the purity of more than 18.25 megohm.cm. According to the kit, the aim of conveniently and quickly detecting the GAPDH gene transcription levels is fulfilled, and the kit has the advantages of high sensitivity, high stability and low experiment cost on the aspect of detecting the gene transcription levels.

Description

technical field [0001] The invention relates to a fluorescent quantitative PCR detection kit for bovine GAPDH gene transcription level. Background technique [0002] Polymerase chain reaction (Polymerase Chain Reaction, PCR) is the most commonly used technology in DNA manipulation technology in vitro. PCR technology puts template DNA, specific primers, dNTPs substrates, heat-resistant DNA polymerase, magnesium ions, etc. in the same buffer reaction system, and performs repeated thermal cycles of high-temperature denaturation, low-temperature annealing, and medium-temperature chain extension to achieve target DNA fragmentation. In the reaction solution, it was 2 n Double amplification (where n is the number of thermal cycles). [0003] Fluorescent quantitative PCR technology is based on flexible PCR reaction, combined with real-time fluorescence detection technology and computer analysis technology. Fluorescence quantitative PCR adds specific fluorescent marker substances ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 裴杰阎萍郭宪包鹏甲梁春年褚敏冯瑞林
Owner LANZHOU INST OF ANIMAL SCI & VETERINARY PHARMA OF CAAS