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PCR premix liquid of fluorescent quantitative PCR thermal cycler and preparation method and application of PCR premix liquid

A fluorescence quantitative, thermal cycler technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., to avoid false positive results, easy identification, and reduce pre-reaction time.

Inactive Publication Date: 2016-08-17
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

SYTO13 has obvious advantages in the application of fluorescent quantitative PCR, but there is no related invention of a master mix suitable for fluorescent quantitative PCR thermal cycler using SYTO13 as a fluorescent marker

Method used

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  • PCR premix liquid of fluorescent quantitative PCR thermal cycler and preparation method and application of PCR premix liquid
  • PCR premix liquid of fluorescent quantitative PCR thermal cycler and preparation method and application of PCR premix liquid
  • PCR premix liquid of fluorescent quantitative PCR thermal cycler and preparation method and application of PCR premix liquid

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preparation example Construction

[0036] The preparation method of described premix is ​​as follows:

[0037] 1) Prepare KCl solution, Tris solution, (NH 4 ) 2 SO 4 solution, MgCl 2 Dissolve, vortex and mix well, store at 4°C for later use;

[0038] 2) Then sterile double distilled water, and the KCl solution prepared in step 1), Tris solution, (NH 4 ) 2 SO 4 solution, and MgCl 2 The solution is pre-mixed in a sterilized storage tube, the pH of the solution is adjusted to 8.5, sterilized after mixing to obtain a pretreatment mixture; the KCl content is 40 μmol to 80 μmol; the (NH 4 ) 2 SO 4 The content is 0-4 μmol; the content of the Tris solution is 10 μmol to 30 μmol; the MgCl 2 The content is 2μmol~4μmol;

[0039] 3) Add 1.8μmol~3μmol dNTPs, 40U~100U DNA polymerase, 5μmol~20μmol dye SYTO13, 30pmol~120pmol reference fluorescent CXR, 0-100μL DMSO and 0-80μL propane to the pretreatment mixture obtained in step 2). Triol, mix well and centrifuge, finally add sterile double distilled water to 1mL, mi...

Embodiment 1

[0042] Add 1 μL of DNA intercalating fluorescent dye SYTO13 (5 mM), 1 μL of reference fluorescent dye CXR (30 μmol), 80 μL of KCl solution (1mol / L), 80 μL of glycerol, 60 μL of DMSO, 10 μL of Tris solution (1mol / L), MgCl 2 Solution (0.1mol / L) 20μL, dNTPs solution 45μL, hot start DNA polymerase (2.5U / μL) added 16μL

[0043] Preparation process:

[0044] Sterilization of equipment and consumables - inorganic compounds KCl, Tris, MgCl 2 Preparation of solution—sterilization of inorganic compound solution (temperature 126°C, time 15min)—cooling storage (4°C)—ingredient 1 (add 200 μL double distilled water, KCl solution, Tris solution, MgCl 2 solution)—high concentration of HCl to adjust the pH of the solution to 8.5——2500r / min vortex for 30s, 4000×g centrifugation for 30s——inorganic compound solution sterilization (temperature 126°C, sterilization 15min)——cooling storage (4°C) ——Ingredient 2 (add dNTPs, DNA polymerase, dye SYTO13, glycerol, DMSO in sequence)——2500r / min vortex fo...

Embodiment 2

[0046] Add 2.4 μL of DNA intercalating fluorescent dye SYTO13 (5 mM), 2 μL of reference fluorescent dye CXR (30 μmol), 80 μL of KCl solution (1mol / L), 80 μL of glycerol, 100 μL of DMSO, 25 μL of Tris solution (1mol / L), MgCl 2 (0.1mol / L) solution 30μL, dNTPs solution 60μL, hot start DNA polymerase (2.5U / μL) added 20μL

[0047] Preparation process:

[0048] Sterilization of equipment and consumables - inorganic compounds KCl, Tris, MgCl 2 Preparation of solution—sterilization of inorganic compound solution (temperature 126°C, time 15min)—cooling storage (4°C)—ingredient 1 (add 200 μL double distilled water, KCl solution, Tris solution, MgCl 2 solution)—high concentration of HCl to adjust the pH of the solution to 8.5——2500r / min vortex for 30s, 4000×g centrifugation for 30s——inorganic compound solution sterilization (temperature 126°C, sterilization 15min)——cooling storage (4°C) ——Ingredient 2 (add dNTPs, DNA polymerase, dye SYTO13, reference fluorescent CXR, glycerol, DMSO in ...

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Abstract

The invention discloses PCR premix liquid of a fluorescent quantitative PCR thermal cycler and a preparation method and application of the PCR premix liquid and belongs to the technical field of biology. According to the PCR premix liquid, DNA double chains are embedded into SYTO13, namely a fluorescence indicator, and dye CXR serves as reference fluorescence. The PCR premix liquid is specially developed for the fluorescent quantitative PCR thermal cycler, and the application effect of the premix liquid in the fluorescent quantitative PCR instrument is stable. SYTO13 serves as the fluorescence indicator, the dye has no inhibition for a PCR reaction, fluorescence intensity is higher, and higher specificity is achieved when the dye is combined with the double chains. The reference fluorescence dye CXR with proper concentration is added, and the PCR premix liquid can be matched with the fluorescent quantitative PCR instrument. Warm start Taq DNA polymerase is selected and used, the PCR experimental period is shortened while a primer is prevented from reacting with a template at normal temperature, and the PCR reaction efficiency and specificity are improved.

Description

technical field [0001] The invention relates to a PCR premix solution of a fluorescent quantitative PCR thermal cycler, a preparation method and application thereof, and belongs to the field of biotechnology. Background technique [0002] Fluorescent quantitative PCR is a technology that adds fluorescent markers to ordinary PCR reactions, collects fluorescent signals during each cycle of annealing or extension of the PCR reaction, monitors the PCR reaction in real time and quantifies the initial template through changes in the fluorescent signal. This technology has many advantages such as strong specificity, high sensitivity, real-time monitoring of reaction process, quantitative analysis of initial template, no need for follow-up operation, low risk of environmental pollution, short analysis time, etc. Wide range of applications. Classified from the labeling method, fluorescent quantitative PCR is mainly divided into two types: dye labeling method and probe labeling metho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q2563/107C12Q2527/125C12Q2521/101
Inventor 刘宁杨艳王桂姬
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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