Gel-based preservation method for polymerase chain reaction agent and reaction reagent

A storage method and chain reaction technology, applied in the field of gel-based polymerase chain reaction reagent storage method and reaction reagents, can solve the problems of large energy consumption, inability to use PCR reagents for long-term storage at room temperature, etc., to reduce storage and transportation costs, The effect of low cost and simple process

Inactive Publication Date: 2013-09-18
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The invention patent "preservation method of polymerase chain reaction mixture (ZL 0011250.4)" discloses a method of using a nucleic acid vacuum concentrator to dry the PCR reaction reagent mixture at low temperature to achieve long-term preservation of the PCR reaction reagent mixture, but this method It requires complex equipment such as special nucleic acid vacuum concentrators or vacuum freeze-dryers, and consumes a lot of energy, which is not a very economical method
The invention patent "PCR and RT-PCR full premix reagents containing high concentration of glycerol (ZL 03117011.0)" discloses a method for long-term storage of PCR reagents at low temperature by adding 30-65% (v / v) glycerol, but the method Cannot be used for long-term storage of PCR reagents at room temperature

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  • Gel-based preservation method for polymerase chain reaction agent and reaction reagent
  • Gel-based preservation method for polymerase chain reaction agent and reaction reagent
  • Gel-based preservation method for polymerase chain reaction agent and reaction reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Analysis of the preservation effect of the PCR reaction reagent mixture after adding the gel

[0034] 1. Add different concentrations and different types of gels to the PCR reaction reagent mixture for storage

[0035] Mix 6048 μL of the mixture of various PCR reaction reagents except the nucleic acid template (including 0.4 μM each of PCR primer F and primer R, 1.4 mM each of dATP, dTTP, dGTP and dCTP, 50 mM KCl, 10 mM Tris / HCl (pH 9.0), MgCl 2 1.5mM, 1%Triton X-100, Taq DNA polymerase 630U) was divided into 14 1.5mL centrifuge tubes at 432μL per tube, and the Add gel-forming substances into each 1.5mL centrifuge tube and mix well, then place the 1.5mL centrifuge tube on a metal bath, and incubate at 50-65°C for 5 minutes according to the dissolution temperature of different gels (lower than The inactivation temperature of Taq DNA polymerase is 100℃), so that the gel is fully dissolved, and then the mixture in each 1.5mL centrifuge tube is divided into 18 ...

Embodiment 2

[0054] Example 2: Analysis of the preservation effect of the PCR reaction reagent mixture after adding the premixed gel

[0055] 1. Add different concentrations and different types of premixed gels to the PCR reaction reagent mixture for storage

[0056] First, prepare a premixed gel twice the concentration in the table according to the concentration and type shown in the first column of Table 3 (14 rows in total); prepare 3024 μL of the mixture of various PCR reaction reagents except the nucleic acid of the sample to be tested (including PCR 0.4 μM each of primer F and primer R, 1.4 mM each of dATP, dTTP, dGTP and dCTP, 50 mM KCl, 10 mM Tris / HCl (pH 9.0), MgCl 2 1.5mM, 1%Triton X-100, Taq DNA polymerase 630U) divided into 14 1.5mL centrifuge tubes according to 216μL per tube; put 2 times the concentration of the premixed gel in the table at the melting point of the gel Melt the gel at high temperature, add 216 μL of melted gel to each of the above 1.5 mL centrifuge tubes (to...

Embodiment 3

[0075] Example 3 Effect of gel-immobilized Taq DNA polymerase on PCR amplification effect after long-term preservation

[0076] 1. Add different concentrations and different types of gels to immobilize and store Taq DNA polymerase

[0077] Add gel-forming substances to 14 1.5mL centrifuge tubes containing 100μL Taq DNA polymerase (2U / μL) according to the concentration and type shown in column 1 of Table 5 (14 rows in total), and mix well. Then place the above 1.5mL centrifuge tubes on a metal bath, and keep warm at 50-63°C for 5 minutes according to the dissolution temperature of different gels to fully dissolve the gel, and then add Taq DNA polymerase in each 1.5mL centrifuge tube The mixture was divided into 18 0.2mL centrifuge tubes according to the amount of 4 μL, and then each group of 18 0.2mL centrifuge tubes containing the mixture were stored at 4°C, 20°C (normal temperature) and 37°C. (Save 6 0.2mL centrifuge tubes containing the mixture under each temperature condit...

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Abstract

The invention relates to a gel-based preservation method for polymerase chain reaction agent and reaction reagent, and the method is realized through the steps that one or more components of polymerase chain reaction liquid is / are embedded in gel for preservation, wherein the melting point of the gel is lower than the inactivation temperature of polymerase used for polymerase chain reaction. In addition, the invention further includes the reaction reagent and a kit both can be stored and transported at normal temperature. The polymerase chain reaction reagent treated by the method disclosed by the invention can be stored and transported at normal temperature, can be used directly without any special treatment. As being in solid state after treatment, the treated polymerase chain reaction reagent mixture can be transported by air conveniently, and the difficulty that the reaction reagent in the prior art can not be transported conveniently for long distance by air as being in liquid state.

Description

technical field [0001] The invention belongs to the technical field of preservation methods for biochemical reagent compositions, and in particular relates to a gel-based preservation method for polymerase chain reaction reagents and reaction reagents. Background technique [0002] In the past 20 years, nucleic acid amplification technology represented by polymerase chain reaction (PCR) has been rapidly developed and applied. Due to the convenience and speed of PCR technology, which can amplify a large number of target nucleic acid fragments in a short period of time, this technology has been widely used in the fields of gene cloning and recombinant expression, biological species identification and classification, species origin and evolution, epidemic pathogen detection and forensic identification. Wide range of applications. Although PCR technology itself has obvious advantages, products based on this technology face some problems in the process of large-scale application...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10C12Q1/68
Inventor 张庆利黄倢杨冰宋晓玲刘庆慧刘莉
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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