Kit and method for extracting microbial genome DNA by magnetic bead method

A microbial and genomic technology, applied in the biological field, can solve problems such as expensive, difficult to obtain, and incomplete cell wall destruction, and achieve the effects of ensuring purity and concentration, promoting hydrogen bond formation, and high extraction efficiency

Inactive Publication Date: 2019-09-27
WUXI NO 2 PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But above these methods have certain shortcomings: 1, the risk of pathogenic microorganisms polluting the environment and infecting staff; Benzyl) is harmful to the environment and staff; 4. Liquid nitrogen is easy to cause frostbite and is not easy to obtain
[0006] At present, the products that can extract various microorganisms including fungal genomic DNA at the same time are mainly American MoBioPowerSoil microbial extraction kit and Omega Fungal DNA extraction kit. Because they have a very large monopoly in the field of microbial genomic DNA extraction, the price is very expensive
Although the price is expensive, the extraction effect is not ideal, and the extraction reagent contains toxic substances, which is easy to cause harm to personnel and the environment

Method used

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  • Kit and method for extracting microbial genome DNA by magnetic bead method
  • Kit and method for extracting microbial genome DNA by magnetic bead method
  • Kit and method for extracting microbial genome DNA by magnetic bead method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Embodiment 1: prepare successively each reagent in the kit of the present invention:

[0067] Bacteriolysis agent II: Prepare 50mM MES by conventional preparation method, and dissolve 0.1M TCEP, 20mM EDTA, 1% TrionX-100 and 1% lysozyme in 50mM MES as solvent. After adjusting the pH to 6.0, the lysate II was obtained.

[0068] Lysis adsorption solution: Prepare 50mM Tris-HCl by conventional preparation method, dissolve 4M guanidine isothiocyanate, 1M sodium chloride, 5mM EDTA, 1% TrionX-100, 2 % triethanolamine dodecylsulfate and 1M thiourea, and adjust the pH to 8.5 to obtain the cleavage adsorption solution.

[0069] Enhanced washing solution: Prepare 50mM Tris-HCl by conventional preparation method, dissolve lithium chloride equivalent to 1M and 60% ethanol in 50mM Tris-HCl as a solvent, and adjust the pH to 6.5 to obtain the enhanced washing solution.

[0070] Microorganism preservation buffer: 10mM Tris-HCL, 100mM NaCL, 5% glycerol, pH7.5.

[0071] Lysate solutio...

Embodiment 2

[0073] Embodiment 2: the DNA extraction of cultivating microorganism

[0074] Extraction was performed using the kit described in Example 1.

[0075] (1) Processing of samples:

[0076] Scrape a certain amount of microorganisms (including Gram-negative bacteria, Gram-positive bacteria and various fungi) with an inoculation loop, add them to 1mL of microorganism preservation buffer, mix well, centrifuge at 12,000rpm for 2 minutes, and discard the supernatant , retain the precipitate.

[0077] (2) Hydrolysis of the cell wall

[0078]2.1 Add 100 μL of lysate I to the precipitate, close the lid, and incubate at 35°C with shaking at 1500 rpm for 30 minutes to obtain the mixed solution I.

[0079] 2.2 Add 100 μL of lysate II, close the lid, and incubate at 35°C with shaking at 1500 rpm for 30 minutes to obtain mixed solution II.

[0080] (3) cracking

[0081] Add 400 μL of Lysis Adsorption Solution and 10 μL of Proteinase K to Mixture II, close the lid, and incubate with shakin...

Embodiment 3

[0091] Bacterolytic agent II is 10 mM MES, 1 mM lytic enzyme enhancer, 5 mM EDTA, 0.01% Triton X-100 and 0.1% lytic enzyme, and the pH of bacteriolytic agent II is 5.5.

[0092] The lysis adsorption solution includes: 10mM Tris-HCl, 0.5M sodium chloride, 1mM EDTA, 0.1% TrionX-100 and nucleic acid separation and absorption accelerator, and the pH of the lysis adsorption solution is 6.0.

[0093] The booster wash solution included 10 mM Tris-HCl, 0.5M LiCl and 50% ethanol, and the pH of the booster wash was 6.5.

[0094] The preparation of all the other solvents is the same as in Example 1.

[0095] Extraction Method:

[0096] Add 50 μL of lysate I to the precipitate, mix and incubate, the mixing time is 10 s, and the incubation time is 10 min, so that the cell wall of Gram-positive bacteria is hydrolyzed to obtain mixture I;

[0097] Add 50 μL of lysate II to the mixture I, mix and incubate, the mixing time is 10 s, and the incubation time is 10 min, so that the fungal cell w...

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Abstract

The invention relates to a kit and method for extracting a microbial genome DNA by a magnetic bead method. The kit comprises a microbial preservation buffer solution, a bacteriolytic agent solution I, proteinase K, silica gel magnetic beads, isopropanol and an ethanol water solution, and further comprises a bacteriolytic agent II, a lysis adsorption solution and a reinforced scrubbing solution. The bacteriolytic agent II comprises MES, EDTA, Triton X-100, lysozyme and a lysozyme enhancer. The lysis adsorption solution comprises Tris-HCl, sodium chloride, EDTA, TrionX-100 and a nucleic acid separation and absorption promoter; and the reinforced scrubbing solution comprises Tris-HCl, lithium chloride and ethanol. By adopting the kit provided by the invention, cell walls of gram-positive bacteria and various fungi can be completely destroyed to form protoplasts, DNA of the protoplast cells can be rapidly released under the action of the lysis adsorption solution, the DNA is efficiently adsorbed on the silica gel magnetic beads, and after the DNA is washed by the scrubbing solution and eluted by an elution solution, microbial DNA with high concentration and purity can be obtained.

Description

technical field [0001] The invention relates to a kit and method for extracting microbial genome DNA by magnetic bead method, in particular to a silica gel magnetic bead extraction method for efficiently extracting genomic DNA of different microorganisms (including Gram-positive bacteria and various fungi) in various samples The kit and method belong to the field of biotechnology. Background technique [0002] Metagenomics is one of the hottest research fields in scientific research in the world today. Metagenome takes the genome of microbial populations in samples as the research object, high-throughput sequencing as the research method, and microbial diversity, population structure, evolutionary relationship, functional activity, mutual cooperation relationship, and relationship with the environment. The purpose of new microbial research methods. Traditional microbiological research is based on isolation and culture, but the existing culture technology can only isolate a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1013C12Q2521/537C12Q2523/308C12Q2527/125C12Q2563/143C12Q2563/149
Inventor 黄学文赵琪安仙园沈兰凤胡仁静
Owner WUXI NO 2 PEOPLES HOSPITAL
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