DNA extracting solution and method for extracting DNA of gram-positive bacteria

A technology of gram-positive bacteria and extract solution, which is applied in the field of DNA extraction and DNA extract solution of gram-positive bacteria, and can solve problems such as health threats to operators, PCR amplification, poor quality, etc.

Pending Publication Date: 2021-07-23
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, its cumbersome operation and time-consuming shortcomings make it difficult to meet the requirements of rapid DNA extraction from a large number of samples. At the same time, toxic and harmful reagents such as chloroform and phenol are used in the extraction process, which poses a threat to the health of operators. Garbage can also pollute the environment
[0003] The bacterial genomic DNA extraction kits currently on the market mainly use lysozyme to break the cell wall, protease to hydrolyze the protein, separate the genomic DNA from the protein, and collect the DNA through a silica gel membrane spin column. The DNA extracted by this kit has high concentration and high quality. Good, and no toxic and harmful reagents such as chloroform are used, but these kits are expensive, contain components that need to be refrigerated, and DNA extraction still takes more than 1 hour, so they are not suitable for DNA extraction of a large number of microbial samples
Subsequently, researchers successively developed methods such as alkaline lysis method and boiling water bath method to quickly extract the genomic DNA of Gram-positive bacteria. Although these methods can extract genomic DNA in a short time, the extracted DNA content is low and the quality is poor. As a template for PCR reactions, it is easy to cause the failure of PCR amplification

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  • DNA extracting solution and method for extracting DNA of gram-positive bacteria
  • DNA extracting solution and method for extracting DNA of gram-positive bacteria

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Embodiment 1

[0030] The extraction of embodiment 1 escherichia coli genomic DNA

[0031] Sufficiently resuspend the Escherichia coli cells in 2 ml of culture with 1 ml of sterile distilled water, centrifuge at 10,000 rpm for 1 min, discard the supernatant to collect the cells, and use solution I (chlorinated Sodium 0.15mol / L, EDTA 10mmol / L, Tris-HCl 15mmol / L with a pH value of 8.5) 450 μL resuspended, standing at 37°C to lyse the cell wall; ethanolamine) 50 μL and mix them upside down, and let stand at room temperature for 10 minutes; add 250 μL of solution III (3mol / L potassium chloride) in an ice bath, centrifuge at 10,000 rpm for 4 minutes after mixing, take the supernatant and transfer it to another test tube, add salicylic acid Methyl ester 500μL, vortex and mix until milky, centrifuge at 11000rpm for 2min, take the upper aqueous phase into another test tube (be careful not to absorb the protein layer and organic phase below), add an equal volume of isopropanol to precipitate for 5min...

Embodiment 2

[0033] The extraction of embodiment 2 Staphylococcus genomic DNA

[0034] Sufficiently resuspend the Staphylococcus cells in 2 ml of the culture with 1 ml of sterile distilled water, centrifuge at 10,000 rpm for 1 min, discard the supernatant to collect the cells, and use solution I (sodium chloride) containing 5 mg / ml lysozyme (added just before use) 0.2mol / L, EDTA20mmol / L, Tris-HCl 20mmol / L with a pH value of 8.5) 450μL resuspended, standing at 37°C to lyse the cell wall; ) 50 μL and mix them upside down, and let stand at room temperature for 12 minutes; add 250 μL of solution III (2.5mol / L potassium chloride) in an ice bath, centrifuge at 11,000 rpm for 3 minutes after mixing, take the supernatant and transfer it to another test tube, add salicylic acid Methyl ester 500μL, vortex and mix until milky, centrifuge at 11000rpm for 2min, take the upper aqueous phase into another test tube (be careful not to absorb the protein layer and organic phase below), add an equal volume o...

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Abstract

The invention discloses a DNA extracting solution and a method for extracting DNA of gram-positive bacteria. The DNA extracting solution consists of a solution I, a solution II, a solution III and a solution IV, wherein the solution I comprises sodium chloride, EDTA and Tris-HCl, and the solution II is triethanolamine lauryl sulfate with the mass volume fraction of 8 to 12.5 percent; the solution III is potassium chloride with the concentration of 2.5 to 3.2 mol / L; the solution IV is methyl salicylate. According to the method, a detergent triethanolamine lauryl sulfate is used for thoroughly cracking cells and denaturating proteins, so that the use of protease K is avoided, and then a potassium chloride solution is added, so that the denatured proteins are precipitated along with generated potassium dodecyl sulfate and are centrifugally removed; methyl salicylate is adopted to replace phenol chloroform isoamyl alcohol to extract the supernate, protein impurities are thoroughly removed, the step of complex water or Tris balance when phenol is used is avoided, and extraction is simpler and more convenient.

Description

technical field [0001] The invention relates to a DNA extraction method, in particular to a DNA extraction solution and a method for extracting Gram-positive bacteria DNA, and belongs to the field of bacterial DNA extraction. Background technique [0002] In the process of identifying Gram-positive bacteria and screening transformants, genomic DNA is often used for PCR amplification. The traditional phenol-chloroform method has the advantages of wide versatility and high stability, so it is widely used. However, its cumbersome operation and time-consuming shortcomings make it difficult to meet the requirements of rapid extraction of DNA from a large number of samples. At the same time, it needs to use toxic and harmful reagents such as chloroform and phenol during the extraction process, which poses a threat to the health of operators. Garbage may also pollute the environment. [0003] The bacterial genomic DNA extraction kits currently on the market mainly use lysozyme to...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/6806
CPCC12N15/1003C12Q1/6806C12Q2527/125
Inventor 许崇敬李凤兰贺付蒙徐永清冯旭
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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