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Fluorogenic quantitative PCR detection kit for bovine ACTB gene transcription level

A detection kit and gene transcription technology, applied in the field of fluorescence quantitative DNA in vitro amplification, can solve problems such as affecting the accuracy of quantification, and achieve highly specific results

Inactive Publication Date: 2013-09-11
LANZHOU INST OF ANIMAL SCI & VETERINARY PHARMA OF CAAS
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

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

Method used

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  • Fluorogenic quantitative PCR detection kit for bovine ACTB gene transcription level
  • Fluorogenic quantitative PCR detection kit for bovine ACTB gene transcription level
  • Fluorogenic quantitative PCR detection kit for bovine ACTB gene transcription level

Examples

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example 1

[0027] 1) Prepare the ACTB fluorescent quantitative PCR reaction solution in proportion, and the 20 μL reaction system is as follows:

[0028] Table 2

[0029]

[0030] Negative control and positive control should be set in the same quantitative reaction.

[0031] 2) Take 1000 μL of 2×SYBR GREEN MIX, 100 μL of primer mixture, and 800 μL of ultrapure water and mix thoroughly to prepare 1900 μL of FQ-PCR reaction master mix.

[0032] 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.

[0033] 4) Prepare 5 tubes of standard ACTB 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.

[0034] 5) FQ-PCR amplification Add 1 μL of the cDNA to be tested (replaced with the above concentrations of standard products here), water or standard ACTB gene template into each react...

example 2

[0038] 1) Prepare the ACTB fluorescent quantitative PCR reaction solution in proportion, and the 20 μL reaction system is as follows:

[0039] table 3

[0040]

[0041] Negative control and positive control should be set in the same quantitative reaction.

[0042] 2) Take 1000 μL of 2×SYBR GREEN MIX, 100 μL of primer mixture, and 800 μL of ultrapure water and mix thoroughly to prepare 1900 μL of FQ-PCR reaction master mix.

[0043] 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.

[0044] 4) Prepare 5 tubes of standard ACTB 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; , prepare 90 tubes of standard ACTB gene template with 10E+4 / mL dilution.

[0045] 5) FQ-PCR amplification. Add 1 μL of diluted standard ACTB gene template (negative control tube plus ultr...

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Abstract

The invention discloses a fluorogenic quantitative PCR detection kit for bovine ACTB gene transcription level. The kit comprises 2XSYBR GREEN MIX, a primer mixed liquor, a standard ACTB genetic template and ultrapure water. The kit of the invention can be used to measure the ACTB gene transcription level accurately, and possesses high specificity (Figure 1). The results of an amplification curve show that ACTB fluorescence signal value accords with a standard "S" typed curve, and the results of a melting curve show that the fluorogenic quantitation possesses high detection specifity. [0]

Description

technical field [0001] The invention relates to a reagent kit which is simple and quick to operate and can quantitatively detect actin-β (β-actin, ACTB) gene transcription levels of different cattle species (including cows, cattle and yaks). The invention belongs to molecular biology Fluorescence quantitative DNA in vitro amplification technology field in detection technology. 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). [000...

Claims

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Application Information

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