Nucleic-acid extraction kit and application thereof

A kit and nucleic acid technology, applied in the field of molecular biology, can solve the problems such as the inability to extract a large amount of mortars, the inability to completely break the cell wall, and the inability to obtain a good wall removal effect, so as to be beneficial to removal, improve the lysis efficiency, and have a good effect. release effect

Inactive Publication Date: 2018-05-04
SUZHOU PRECISION BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the mechanical method mainly uses liquid nitrogen freeze-drying to grind and break the cell wall, but this method cannot completely break the cell wall, and most of this method uses toxic organic solvents, phenol, etc., and the operation takes a long time, and it

Method used

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  • Nucleic-acid extraction kit and application thereof
  • Nucleic-acid extraction kit and application thereof
  • Nucleic-acid extraction kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0054] Example 1 Preparation of detection kit

[0055] (1) Preparation of reagents

[0056] Buffer A: 0.5-0.5M Tris-Cl buffer (pH5.0-8.0), 0.5-1.5M NaCl;

[0057] Enzyme solution B: 0.05-0.2M phosphate buffer (pH6.8-7.4), 0.25M MgCl2, 80-120mg / mL snail enzyme;

[0058] Lysis Solution C: 0.05-0.5M Tris-Cl buffer (pH 5.0-8.0), 0.05-0.2M EDTA, 8-12% SDS, 8-12% Tween-40;

[0059] Suspension D: 5-40mg / ml RNAaseA

[0060] Remover E: 0.2-1M Al2(SO4)3, 80-350g / L CTAB, 0.5-1.5M NaCl;

[0061] Binding solution F: 0.1-0.5M Tris-Cl buffer (pH5.0-8.0), 2-8M guanidine isothiocyanate;

[0062] Rinsing solution G: 0.1-0.5M Tris-Cl buffer (pH 5.0-8.0), 15-25% (volume ratio) absolute ethanol;

[0063] Eluent H: sterile deionized water;

[0064] (2) Consumables

[0065] Glass beads, adsorption column and collection tube

[0066] (3) Assembly of the kit

[0067] The primer composition and the detection probe are assembled with the reagents related to the kit to prepare the detection kit.

Example Embodiment

[0068] Example 2 Extraction of sample DNA (A1 / B1 / C1)

[0069] Select 3 stool samples: A, B, C, and divide them into 6 evenly, labeled: A1-A6, B1-B6, C1-C6, A1, B1, and C1. Three samples are processed by the method of this embodiment. The specific The DNA extraction method includes the following steps:

[0070] (1) Take 800μl buffer A and 200mg glass beads into a 2ml centrifuge tube;

[0071] (2) Add 200mg sample to the above 2ml centrifuge tube, vortex and mix for 30s;

[0072] (3) Add 100μl of enzyme solution B to the centrifuge tube of step (2), and incubate at 45-65°C for 20 minutes;

[0073] (4) Add 50μl of lysate C to the sample, vortex for 5min to mix the sample, 12,000rpm (~13,400×g), centrifuge for 60s, transfer the supernatant (400μl) to a new 2ml centrifuge tube;

[0074] (5) Add 300μl of suspension D, vortex for 5-10s, place at room temperature for 5min, 12,000rpm (~13,400×g), centrifuge for 60s, and precipitate sample particles;

[0075] (6) Transfer the supernatant to a new ...

Example Embodiment

[0084] Example 3 Extraction of sample DNA (A1 / B1 / C1)

[0085] The three samples A1, B1, and C1 are processed by the method of this embodiment. The specific DNA extraction method includes the following steps:

[0086] (1) Take 800μl buffer A and 100mg glass beads into a 2ml centrifuge tube;

[0087] (2) Add 200mg sample to the above 2ml centrifuge tube, vortex and mix for 30s;

[0088] (3) Add 50μl of enzyme solution B to the centrifuge tube of step (2), and incubate at 40-50°C for 120min;

[0089] (4) Add 100μl of lysate C to the sample, vortex for 5min to mix the sample, 12,000rpm (~13,400×g), centrifuge for 60s, transfer the supernatant (400μl) to a new 2ml centrifuge tube;

[0090] (5) Add 300μl of suspension D, vortex for 5-10s, place at room temperature for 5min, 12,000rpm (~13,400×g), centrifuge for 60s, and precipitate sample particles;

[0091] (6) Transfer the supernatant to a new 2ml centrifuge tube, add 50μl of remover E to mix, and place at 4°C for 2 minutes;

[0092] (7) Centr...

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PUM

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Abstract

The invention belongs to the field of biology and relates to a nucleic-acid extraction kit and application thereof. The kit comprises glass beads, helicase, lysis solution and a remover, wherein the lysis solution comprises Tween-40, and the remover comprises CTAB and Al2(SO4)3. The kit has the advantages that fungal cell walls can be broken by high efficiency, and high-quality fungal nucleic acidcan be obtained in a fast and high-efficiency manner.

Description

technical field [0001] The invention belongs to the field of molecular biology and relates to a nucleic acid extraction kit and its application, in particular to a nucleic acid extraction kit for fungi and its application. Background technique [0002] Fungi are a large class of eukaryotic cell-type microorganisms. It is widely distributed in nature and has a wide variety. At present, there are 10,000 genera and more than 100,000 species. Most of them are beneficial to humans, while a few are harmful to humans and can cause diseases of humans, animals and plants. [0003] There are more than 400 species of fungi related to medicine, and 50-100 species are common, which can cause infectious, toxic and hypersensitivity diseases in humans. Such as: skin ringworm, tinea versicolor, pneumonia, enteritis, allergic dermatitis, etc. [0004] At present, the detection methods of common fungi in clinical practice are mainly direct microscopic examination and isolation culture metho...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12N1/06C12Q1/6806C12Q1/04
CPCC12N1/06C12N15/1006C12Q1/6806C12Q2563/149
Inventor 穆延召朱永亮周燕
Owner SUZHOU PRECISION BIOTECH CO LTD
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