Rapid extraction method of DNA of gram positive bacterial genome
A Gram-positive, extraction method technology, which is applied in the field of rapid extraction of G+ Gram-positive bacterial genomic DNA, can solve the problems of difficult to break the cell wall, long time required, and expensive price of lysozyme, etc., and achieve simple technical operation , cost-saving effect
Pending Publication Date: 2020-01-03
NORTHWEST A & F UNIV
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Problems solved by technology
In addition, there are still a large amount of special component teichoic acid, which makes the cell wall extremely difficult to break
At present, in the process of conventional G+ bacterial genomic DNA extraction, lysozyme is used to break the cell wall, and most of them use lysozyme with a final concentration of 300ug / ml. It takes 1 hour to incubate on ice, which is too long
Moreover, lysozyme is more expensive and requires more attention in transportation and storage, which greatly increases the cost of G+ bacterial genomic DNA extraction, and the chemical essence of lysozyme is protein, which breaks the cell wall and introduces new proteins at the same time. As a result, the extracted genomic DNA is impure, which affects the subsequent detection results, and also puts forward higher requirements for protein extraction technology in the genome extraction process, increasing test time and cost
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[0020] Rapid extraction method of G+ gram-positive bacteria genomic DNA
[0021] 1) Separation and collection of G+ bacteria: Take sterile milk, add a known amount of Gram-positive bacteria, and centrifuge at 9000 r / min for 2 minutes to collect the bacteria.
[0022] 2) Disruption of G+ bacterial cell wall: Use 300 microliters of TENT buffer (10mM Tris-HCl, 0.1M NaCl, 1mM EDTA, 5%[v / v]Triton X100, pH 8.0) to resuspend the bacteria, and boil for three minutes at 100°C , Centrifuge at 3000g for 5min, and collect the supernatant.
[0023] 3) Precipitation and washing of G+ bacterial genome: add 95% ethanol to the supernatant, place it at -20°C for 20 minutes, centrifuge at 12000g for 10 minutes, and collect the supernatant. Add 75% ethanol to the supernatant, mix upside down, and centrifuge at 12000g for 5 minutes. Repeat 2-3 times, collect the precipitate, add sterile ddH 2 O, dissolve the DNA, use the OD260 / OD280 method to detect the quality of the extracted DNA. Store at -20°C tem...
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The invention discloses a rapid extraction method of DNA of gram positive bacterial genome. The rapid extraction method includes the following steps that G+ bacteria are collected by centrifugation, and centrifugation is performed for 2min under 9,000r / min; G+ bacteria cell walls are broken by using pyrolysis buffer-TENT buffer, then boiling is performed at 100 DEG C for three minutes, and 3,000gcentrifugation is performed for 5min to collect supernatant; the precipitation of G+ bacterial genome is performed, the method for collecting G+ bacterial genome comprises the steps that 75% ethanol is added to the supernatant, the mixture is evenly mixed in an upside down mode, and 12,000g centrifugation is performed for 5min; the steps are repeated for 2-3 times, precipitate is collected, and sterile ddH20 is added to dissolve the DNA; and the mixture is stored at -20 DEG C for standby use. The technology is simple in operation, time is saved, introduction of protein contamination is avoided, the cost is saved, and the rapid extraction method is suitable for popularization and application.
Description
Technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for rapidly extracting genomic DNA of gram-positive bacteria, and specifically, to a method for quickly extracting genomic DNA of G+ (gram-positive) gram-positive bacteria. Background technique [0002] Common Gram-positive bacteria include: Staphylococcus, Streptococcus, Pneumococcus, Bacillus anthracis, Bacillus diphtheria, Bacillus tetanus, etc. Moreover, most pyogenic cocci belong to Gram-positive bacteria, which can produce exotoxin to cause disease in humans and animals. Gram-positive bacteria have thicker cell walls, mainly composed of peptidoglycan and acidic polysaccharides including teichoic acid, about 20-80 nm. Peptidoglycan is rich in content, with 15-50 layers, each with a thickness of 1nm, accounting for about 50-80% of the dry cell weight. In addition, there are a large number of special components teichoic acid, which makes the cell wall extremely difficult to br...
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CPCC12N15/1003
Inventor 陈德坤程红玉管雄马文涛
Owner NORTHWEST A & F UNIV