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Genome extraction method for improving DNA quality of myxobacteria by virtue of pretreatment

An extraction method, the technology of myxobacteria, which is applied in the field of molecular biology, can solve the problems of low purity of total DNA of myxobacteria, instability of DNA extracts, and easy aggregation of cells, etc., to achieve easy realization and scale-up production, complete DNA release, and lysis good effect

Inactive Publication Date: 2015-04-08
SHAANXI UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, S. cellulosus can secrete a large amount of exopolysaccharide mucus during growth, which makes the cells easy to aggregate and difficult to disperse, which is not conducive to subsequent molecular operations, such as genomic DNA extraction, genomic cloning, genomic library construction, and Southern hybridization.
The total DNA purity of myxobacteria extracted by the general genome extraction method in the prior art is low, and the DNA extract is very unstable

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] 1) Pretreatment

[0035] Cultivate myxobacteria on CNST medium with 3-4 pieces of sterilized filter paper with a width of 1.5-2cm at 28°C for 7 days, then transfer 0.3g of the bacteria to a 2.0mL centrifuge tube, and transfer to the centrifuge tube Add 1.0 mL of sterile water to wash the cells, centrifuge at 6000 r / min for 5 min, discard the supernatant, and keep the cells; add 300 μL of 0.3% sucrose monoester S-1570 aqueous solution to the cells, Then oscillate with an ultrasonic oscillator for 5 minutes to disperse the bacteria evenly; wherein, the strain of myxobacteria is S. cellulosus ATCC25532

[0036] 2) Cell Lysis

[0037] Add 500 μL of DNA extraction buffer and 25 μL of proteinase k solution with a concentration of 20 mg / L to the shaken centrifuge tube, and heat in a water bath at 65°C for 1 hour. Wherein, the extraction buffer includes Tris-HCl with a pH value of 8.0 and 10 mmol / L; EDTA with a pH value of 8.0 and 5 mmol / L; SDS with a mass concentration of 5%...

Embodiment 2

[0043] 1) Pretreatment

[0044] Cultivate myxobacteria on CNST medium with 3-4 pieces of sterilized filter paper with a width of 1.5-2cm at 30°C for 5 days, then transfer 0.4g of the bacteria to a 2.0mL centrifuge tube, and transfer to the centrifuge tube Add 1.0mL sterile water to wash the cells, centrifuge at 6000r / min for 5min, discard the supernatant, and keep the cells; add 500μL of 0.4% sucrose monoester S-1570 aqueous solution to the cells, and use Vibrate for 10 min with an ultrasonic oscillator to disperse the cells evenly.

[0045] 2) Cell Lysis

[0046] Add 600 μL of DNA extraction buffer and 28 μL of proteinase k solution with a concentration of 23 mg / L to the shaken centrifuge tube, and heat in a water bath at 70° C. for 50 min. Wherein, the extraction buffer includes Tris-HCl with a pH value of 8.0 and 10 mmol / L; EDTA with a pH value of 8.0 and 15 mmol / L; SDS with a mass concentration of 5%, and the balance is water.

[0047] 3) Remove protein

[0048] Add 75...

Embodiment 3

[0052] 1) Pretreatment

[0053] Cultivate myxobacteria on CNST medium with 3-4 pieces of sterilized filter paper with a width of 1.5-2cm at 29°C for 6 days, then transfer 0.5g of the bacteria to a 2.0mL centrifuge tube, and transfer to the centrifuge tube Add 1.0mL of sterile water to wash the cells, centrifuge at 6000r / min for 5min, discard the supernatant, and keep the cells; add 300μL of 0.5% Tween 80 aqueous solution to the cells, and then use ultrasonic Oscillate for 8 minutes to make the cells evenly dispersed.

[0054] 2) Cell Lysis

[0055] Add 750 μL of DNA extraction buffer (Tris-HCl with a pH value of 8.0 and 15 mmol / L; EDTA with a pH value of 8.0 and 2 mmol / L; SDS with a mass concentration of 10%) and a concentration of 20 mg in the shaken centrifuge tube. / L proteinase k solution 30μL, heated in a water bath at 68°C for 30min.

[0056] 3) Remove protein

[0057] Add 1000 μL of the mixture of chloroform and isoamyl alcohol (the volume ratio of chloroform to iso...

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Abstract

The invention provides a genome extraction method for improving DNA quality of myxobacteria by virtue of pretreatment. The method comprises the following steps: in light of a phenomenon that in the culture process of myxobacteria, a lot of mucoid exopolysaccharides are generated to wrap the bacterium, so that cells in the liquid are hard to disperse, adding a nonionic surfactant into washed myxobacteria during pretreatment and oscillating by using an ultrasonic oscillator; and then, adding a SDS gene extraction buffer liquid and protease k for water bath so as to further uniformly mix the surfactant in the liquid, so that the cells are fully split. The method provided by the invention is integrally fast and simple to operate, and reagents and experimental instruments used are common instruments and reagents for a laboratory, so that the method is suitable for common and conventional experimental operations. The method is wide in application range, suitable for genome DNA extraction of all myxobacteria and strong in purifying capacity. The obtained DAN can be used for satisfying operations such as subsequent PCR amplification and DNA sequencing.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a method for extracting genome DNA of myxobacteria, in particular to a genome extraction method for improving DNA quality of myxobacteria through pretreatment. Background technique [0002] Myxobacteria (myxobacteria) are Gram-negative bacilli capable of gliding, mainly found in humus, herbivorous animal feces, soil and bark tissue, with complex multicellular sociological behaviors that can form a variety of diverse, functional Diverse bioactive substances. Although myxobacteria are prokaryotes, they have many physiological and biochemical characteristics similar to eukaryotes, such as the similar conduction mode between cells to regulate their movement and fruiting process. According to "differences in substrate types", myxobacteria can be divided into two categories: lytic bacteria and cellulolytic groups. The former can lyse complete living cells of microorganisms such as yeast...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 龚国利张甜
Owner SHAANXI UNIV OF SCI & TECH
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