CN101235379B - Method for extracting bacteria plasmid DNA - Google Patents

Method for extracting bacteria plasmid DNA Download PDF

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CN101235379B
CN101235379B CN200710010272XA CN200710010272A CN101235379B CN 101235379 B CN101235379 B CN 101235379B CN 200710010272X A CN200710010272X A CN 200710010272XA CN 200710010272 A CN200710010272 A CN 200710010272A CN 101235379 B CN101235379 B CN 101235379B
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solution
plasmid dna
bacterium
add
centrifugal
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CN101235379A (en
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郑乐
刘宛
李培军
张利红
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Institute of Applied Ecology of CAS
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Institute of Applied Ecology of CAS
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Abstract

The invention relates to molecular biology, which particularly relates to an extracting method of bacterial plasmid DNA. The extracting method comprises the following steps: jolting and overnight culturing microbial thallus in fluid medium under the temperature which is 25 DEG C-30 DEG C, adding buffer solution in bacterial liquid which is overnight cultured, centrifuging to gather and wash to get cracking thallus, separating and purifying plasmid DNA, directly using plasmid DNA which is got in various molecular biology tests such as restriction enzyme, PCR and the like. The extracting method has the advantages of simple and convenient operation and high quality, and meanwhile, the invention is suitable for the extraction of Gram-negative bacteria and Gram-negative bacteria plasmid DNA, therefore, the extracting method has wide adaptability.

Description

The method that a kind of bacteria plasmid DNA extracts
Technical field
The present invention relates to molecular biology, specifically a kind of extracting method of bacteria plasmid DNA.
Background technology
Plasmid is the circular double stranded DNA molecule that is present in the independently duplicated and genetic stability of the outer energy of bacterial chromosome, and plasmid DNA is engineered common carrier, can carry foreign gene and enter and increase in bacterium, zooblast or the plant materials and express.Efficient that plasmid DNA is extracted and quality have direct relation to the success or not of subsequent experimental (enzyme is cut, pcr amplification etc.), therefore extract from bacterial cell quickly and efficiently and plasmid purification has great importance.
From bacterium, extract the normal method that adopts of plasmid DNA at present boiling method, SDS method, alkaline lysis etc. are arranged, these methods all are to extract at the intestinal bacteria that have artificial constructed plasmid usually, length consuming time, quality are low, and use toxic substances such as phenol, chloroform in the leaching process, not only contaminate environment but also be detrimental to health.For a lot of organic pollutant degradation bacteriums, the characteristic of contaminant degradation all is by the interior living plasmid regulation and control of bacterium or relevant with plasmid, see clearly the bacterium for degrading plasmid and in gene on the plasmid and the relation between the organic pollutant degradation, to help more profoundly to understand inherent mechanism and the process of microorganism to organic pollutant degradation, be the basis of carrying out this research so seek a kind of safe and efficient, easy plasmid extracting method that also can be applied to multiple organic pollutant degradation bacterium fast.
Summary of the invention
The method that the object of the present invention is to provide a kind of safe, efficient, suitable various bacteria plasmid DNA to extract.
The method that the plasmid DNA of organic pollutant degradation bacterium is extracted.
For achieving the above object, the technical solution used in the present invention is:
The method of extracting:
1) cultivation of bacterium: use in the liquid nutrient medium the single bacterium colony of the bacterium of picking always 25-30 ℃ of jolting incubated overnight on bacterium;
2) collection of thalline, cracking: when being the Gram-negative bacterium, get the bacterium liquid that 1.5-2.0mL cultivated 16-24 hour, and under 10000-12000rpm centrifugal 30-60 second, collect thalline; With 1-1.5mL STE solution washing twice, centrifugal 30 seconds of 10000rpm; Then in bacterial precipitation, add 100-300 μ L solution I, add 200-600 μ L solution II again, ice bath 3-5 minute, get cracking bacterium liquid;
When being Gram-positive bacterium when cultivating respectively, get the bacterium liquid that 3.0-4.0mL cultivated 10-16 hour, and under 8000-10000rpm centrifugal 60-90 second, collect thalline; With 2.0-3.0mL STE solution washing twice, centrifugal 30 seconds of 10000rpm; Then in bacterial precipitation, add 100-200 μ L solution 1 and 50-100 μ L N,O-Diacetylmuramidase, 37 ℃ water-bath 30-60 minute, and then add 200-600 μ L solution II, ice bath 3-5 minute, cracking bacterium liquid;
3) renaturation of plasmid DNA, separate with chromosomal DNA: in step 2) in add 150-400 μ L solution III in the cracking bacterium liquid that obtains, ice bath 15-20 minute, under 4 ℃ centrifugal 10 minutes, get supernatant with 12000rpm; And in supernatant liquor, add Virahol isopyknic with it, and room temperature was placed 5 minutes, and under 4 ℃ with 12000rpm centrifugal 5 minutes again, it was stand-by to get its precipitation;
4) precipitation of plasmid DNA: the precipitation in the step 3) is dissolved with the aseptic redistilled water of 100-400 μ L, and the 7.5molL of 1/2 volume of the sterilized water of adding dissolution precipitation -1NH 4Ac and 5molL -1LiCl ice bath 3-5 minute, under 4 ℃ with 12000rpm centrifugal 5 minutes, gets supernatant; And in supernatant liquor, add Virahol isopyknic with it, and room temperature was placed 40 minutes, and precipitation is plasmid DNA;
Wherein: solution I is a 50mmol/L glucose, 25mmol/L TrisCl, pH8.0 and 10mmol/L EDTA, pH8.0; Solution II is 0.2mol/L NaOH and 1%SDS; Solution III is that potassic concentration is KAc and the 5mol/L acetate moiety of 3-5mol/L in the solution, pH4.8; NH 4Molar ratio between Ac and the LiCl is 1: 1.
The composition of described bacterium liquid nutrient medium commonly used is: glucose 1.0g, yeast extract paste 1.0g, MgSO 47H 2O 0.2g, Na 2CO 30.1g, CaCl 22H 2O 0.01g, MnSO 4H 2O 0.02g, FeSO 47H 2O 0.005g, NH 4NO 31.0g, KH 2PO 40.4g, Na 2HPO 40.6g adding distil water is to 1L, pH7.2-7.5.
The above-mentioned plasmid DNA that obtains can be used washing with alcohol, adds 50-100 μ L TE dissolving behind the air drying ,-20 ℃ of preservations.
The washing of described plasmid DNA washing with alcohol 2-4 time, wherein concentration of ethanol is 60-80%.
The composition of STE solution is 10mmol/L TrisCl, pH8.0,10mmol/L NaCl and 1mmol/LEDTA, pH8.0; The TE damping fluid is 10mmol/L TrisHCl, pH8.0 and 1mmol/L EDTA, pH8.0; Described N,O-Diacetylmuramidase (Lysozyme Egg White, concentration is 50mg/mL)
The advantage that the present invention had:
1. easy and simple to handle, safety.The used instrument of the present invention is simple, and is easy to operate.Use NH 4Ac replaces phenol, chloroform to remove albumen, can effectively reduce the harm to human body, and reduces the pollution to environment.
2. quality height.The bacterium liquid of selecting suitable condition to carry out microbial culture and suitable volumes carries out plasmid DNA and extracts, and can improve the efficient of extraction to greatest extent; Utilize LiCl to handle precipitated rna in a large number simultaneously, and reduce the digestion time of RNaseA; Replace 2 times of volume of ethanol to precipitate plasmid DNA with the equal-volume Virahol, can obtain better sedimentation effect, the A of the final plasmid DNA that obtains 260/ A 280All reached 1.80, can satisfy the requirement of conventional molecular biology experiment fully.
3. applied range.In having the organic pollutant degradation bacterium of plasmid, existing gram-negative bacterium also has gram-positive bacterium, the experiment proved that the present invention is not only applicable to the extraction of gram-negative bacterium plasmid DNA, be equally applicable to gram-positive bacterium, therefore have extensive applicability.
Description of drawings
Fig. 1 is the horizontal agarose gel electrophoresis spectrogram of DNA; 1 swimming lane wherein is 1kb DNAmarker, and 5 swimming lanes are λ DNA/HindIII marker, and middle three swimming lanes difference 2 is Gordon Salmonella ZCG2, and 3 is pseudomonas B61, and 4 is human pallid bacillus ZHL4.
Embodiment
The present invention is further described below in conjunction with embodiment.
Embodiment 1
Will be at Gordon Salmonella ZCG2 of slant culture, its single bacterium colony of picking 25 ℃ of shaking culture in bacterium liquid nutrient medium commonly used were got 3.0mL bacterium liquid after 12 hours, and centrifugal 70 seconds of 9000rpm is with 2.0mLSTE solution washing twice, centrifugal 30 seconds of 10000rpm; Add earlier 150 μ L solution I and 50 μ L N,O-Diacetylmuramidases (available from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd), 37 ℃ of water-baths 40 minutes, and then add 400 μ L solution II, ice bath 5 minutes; Add 300 μ L solution III, ice bath 20 minutes, 4 ℃, centrifugal 10 minutes of 12000rpm; Get supernatant, add the equal-volume Virahol, room temperature was placed 5 minutes, and 4 ℃, centrifugal 5 minutes of 12000rpm; With the aseptic redistilled water dissolution precipitation of 300 μ L, add the 7.5molL of 150 μ L -1NH 4Ac and 5molL -1LiCl, ice bath 5 minutes, 4 ℃, centrifugal 5 minutes of 12000rpm; Get supernatant, and add Virahol isopyknic with it in supernatant liquor, room temperature was placed 40 minutes, and precipitation is plasmid DNA; Above-mentioned precipitation with 70% washing with alcohol twice, is added 70 μ L TE dissolving behind the air drying.
Wherein: the composition of bacterium liquid nutrient medium commonly used is: glucose 1.0g, yeast extract paste 1.0g, MgSO 47H 2O 0.2g, Na 2CO 30.1g, CaCl 22H 2O 0.01g, MnSO 4H 2O 0.02g, FeSO 47H 2O 0.005g, NH 4NO 31.0g, KH 2PO 40.4g, Na 2HPO 40.6g adding distil water is to 1L, pH7.2,121 ℃ of sterilization 30min; Solution I is a 50mmol/L glucose, 25mmol/LTrisCl, pH8.0 and 10mmol/L EDTA, pH8.0; Solution II is 0.2mol/L NaOH and 1%SDS; Solution III is that potassic concentration is KAc and the 5mol/L acetate moiety of 5mol/L in the solution, pH4.8; NH 4Molar ratio between Ac and the LiCl is 1: 1.The composition of STE solution is 10mmol/LTrisCl, pH8.0,10mmol/L NaCl and 1mmol/L EDTA, pH8.0; The TE damping fluid is 10mmol/L TrisHCl, pH8.0 and 1mmol/L EDTA, pH8.0; The extraction result of its plasmid DNA (referring to Fig. 1 the 2nd swimming lane), the content that extracts DNA is 28.9 μ g/mL, A 260/ A 280=1.80.
Embodiment 2
Will be at the pseudomonas B61 of slant culture, its single bacterium colony of picking 28 ℃ of shaking culture in bacterium liquid nutrient medium commonly used were got 2.0mL bacterium liquid after 18 hours, and centrifugal 45 seconds of 10000rpm is with 1.5mL STE solution washing twice, centrifugal 30 seconds of 10000rpm; Add 135 μ L solution I earlier, and then add 270 μ L solution II, ice bath 4 minutes; Add 200 μ L solution III, ice bath 20 minutes, 4 ℃, centrifugal 10 minutes of 12000rpm; Get supernatant, add the equal-volume Virahol, room temperature was placed 5 minutes, and 4 ℃, centrifugal 5 minutes of 12000rpm; With the aseptic redistilled water dissolution precipitation of 200 μ L, add the 7.5molL of 100 μ L -1NH 4Ac and 5molL -1LiCl, ice bath 5 minutes, 4 ℃, centrifugal 5 minutes of 12000rpm; Get supernatant, add isopyknic Virahol, room temperature was placed 40 minutes, and precipitation is plasmid DNA; Above-mentioned precipitation with 70% washing with alcohol twice, is added 60 μ LTE dissolving behind the air drying.
Wherein: the composition of bacterium liquid nutrient medium commonly used is: glucose 1.0g, yeast extract paste 1.0g, MgSO 47H 2O 0.2g, Na 2CO 30.1g, CaCl 22H 2O 0.01g, MnSO 4H 2O 0.02g, FeSO 47H 2O 0.005g, NH 4NO 31.0g, KH 2PO 40.4g, Na 2HPO 40.6g adding distil water is to 1L, pH7.4,121 ℃ of sterilization 30min; Solution I is a 50mmol/L glucose, 25mmol/LTrisCl, pH8.0 and 10mmol/L EDTA, pH8.0; Solution II is 0.2mol/L NaOH and 1%SDS; Solution III is that potassic concentration is KAc and the 5mol/L acetate moiety of 4mol/L in the solution, pH4.8; NH 4Molar ratio between Ac and the LiCl is 1: 1.The composition of STE solution is 10mmol/LTrisCl, pH8.0,10mmol/L NaCl and 1mmol/L EDTA, pH8.0; The TE damping fluid is 10mmol/L TrisHCl, pH8.0 and 1mmol/L EDTA, pH8.0; The extraction result of its plasmid DNA (referring to Fig. 1 the 3rd swimming lane), the content that extracts DNA is 42.3 μ g/mL, A 260/ A 280=1.85.
Embodiment 3
Will be at the human pallid bacillus ZHL4 of slant culture, its single bacterium colony of picking 30 ℃ of shaking culture in bacterium liquid nutrient medium commonly used were got 1.5mL bacterium liquid after 20 hours, and centrifugal 35 seconds of 11000rpm is with 1.0mLSTE solution washing twice, centrifugal 30 seconds of 10000rpm; Add 100 μ L solution I earlier, and then add 200 μ L solution II, ice bath 3 minutes; Add 150 μ L solution III, ice bath 15 minutes, 4 ℃, centrifugal 10 minutes of 12000rpm; Get supernatant, add the equal-volume Virahol, room temperature was placed 5 minutes, and 4 ℃, centrifugal 5 minutes of 12000rpm; With the aseptic redistilled water dissolution precipitation of 200 μ L, add the 7.5molL of 100 μ L1 -1NH 4Ac and 5molL -1LiCl, ice bath 5 minutes, 4 ℃, centrifugal 5 minutes of 12000rpm; Get supernatant, add isopyknic Virahol, room temperature was placed 40 minutes, and precipitation is plasmid DNA; Above-mentioned precipitation with 70% washing with alcohol twice, is added 50 μ L TE dissolving behind the air drying.
Wherein: the composition of bacterium liquid nutrient medium commonly used is: glucose 1.0g, yeast extract paste 1.0g, MgSO 47H 2O 0.2g, Na 2CO 30.1g, CaCl 22H 2O 0.01g, MnSO 4H 2O 0.02g, FeSO 47H 2O 0.005g, NH 4NO 31.0g, KH 2PO 40.4g, Na 2HPO 40.6g adding distil water is to 1L, pH7.3,121 ℃ of sterilization 30min; Solution I is a 50mmol/L glucose, 25mmol/LTrisCl, pH8.0 and 10mmol/L EDTA, pH8.0; Solution II is 0.2mol/L NaOH and 1%SDS; Solution III is that potassic concentration is KAc and the 5mol/L acetate moiety of 3mol/L in the solution, pH4.8; NH 4Molar ratio between Ac and the LiCl is 1: 1.The composition of STE solution is 10mmol/LTrisCl, pH8.0,10mmol/L NaCl and 1mmol/L EDTA, pH8.0; The TE damping fluid is 10mmol/L TrisHCl, pH8.0 and 1mmol/L EDTA, pH8.0; The extraction result of its plasmid DNA (referring to Fig. 1 the 4th swimming lane), the content that extracts DNA is 35.3 μ g/mL, A 260/ A 280=1.82.

Claims (3)

1. the method extracted of a bacteria plasmid DNA is characterized in that:
1) cultivation of bacterium: use in the liquid nutrient medium the single bacterium colony of the bacterium of picking always 25-30 ℃ of jolting incubated overnight on bacterium; The composition of described bacterium liquid nutrient medium commonly used is: glucose 1.0g, yeast extract paste 1.0g, MgSO 47H 2O 0.2g, Na 2CO 30.1g, CaCl 22H 2O 0.01g, MnSO 4H 2O 0.02g, FeSO 47H 2O 0.005g, NH 4NO 31.0g, KH 2PO 40.4g, Na 2HPO 40.6g adding distil water is to 1L, pH7.2-7.5;
2) collection of thalline, cracking: when bacterium is the Gram-negative bacterium, get cultivation 16-24 hour the bacterium liquid that the 1.5-2.0mL step 1) obtains, and under 10000-12000rpm centrifugal 30-60 second, collect thalline; With 1-1.5mL STE solution washing twice, centrifugal 30 seconds of 10000rpm; Then in bacterial precipitation, add 100-300 μ L solution I, add 200-600 μ L solution II again, ice bath 3-5 minute, get cracking bacterium liquid;
When being Gram-positive bacterium when cultivating respectively, get cultivation 10-16 hour the bacterium liquid that the 3.0-4.0mL step 1) obtains, and under 8000-10000rpm centrifugal 60-90 second, collect thalline; With 2.0-3.0mL STE solution washing twice, centrifugal 30 seconds of 10000rpm; Then in bacterial precipitation, add 100-200 μ L solution I and 50-100 μ L N,O-Diacetylmuramidase, 37 ℃ water-bath 30-60 minute, and then add 200-600 μ L solution II, ice bath 3-5 minute, cracking bacterium liquid; The composition of STE solution is 10mmol/L TrisCl, pH8.0,10mmol/L NaCl and 1mmol/L EDTA, pH8.0;
3) renaturation of plasmid DNA, separate with chromosomal DNA: in step 2) in add 150-400 μ L solution III in the cracking bacterium liquid that obtains, ice bath 15-20 minute, under 4 ℃ centrifugal 10 minutes, get supernatant with 12000rpm; And in supernatant liquor, add Virahol isopyknic with it, and room temperature was placed 5 minutes, and under 4 ℃ with 12000rpm centrifugal 5 minutes again, it was stand-by to get its precipitation;
4) precipitation of plasmid DNA: the precipitation that step 3) obtains is dissolved with the aseptic redistilled water of 100-400 μ L, and the 7.5molL of 1/2 volume of the sterilized water of adding dissolution precipitation -1NH 4Ac and 5molL -1LiCl ice bath 3-5 minute, under 4 ℃ with 12000rpm centrifugal 5 minutes, gets supernatant; And in supernatant liquor, add Virahol isopyknic with it, and room temperature was placed 40 minutes, and precipitation is plasmid DNA;
Wherein: solution I is a 50mmol/L glucose, 25mmol/L TrisCl, pH8.0 and 10mmol/L EDTA, pH8.0; Solution II is 0.2mol/L NaOH and 1%SDS; Solution III is that potassic concentration is KAc and the 5mol/L acetate moiety of 3-5mol/L in the solution, pH4.8; Molar ratio between NH4Ac and the LiCl is 1: 1.
2. by the method for the described bacteria plasmid DNA extraction of claim 1, it is characterized in that: the above-mentioned plasmid DNA washing with alcohol that obtains adds 50-100 μ L TE dissolving behind the air drying,-20 ℃ of preservations, the TE damping fluid is 10mmol/L TrisHCl, pH8.0 and 1mmol/L EDTA, pH8.0.
3. by the method for the described bacteria plasmid DNA extraction of claim 2, it is characterized in that: the washing of described plasmid DNA washing with alcohol 2-4 time, wherein concentration of ethanol is 60-80%.
CN200710010272XA 2007-02-02 2007-02-02 Method for extracting bacteria plasmid DNA Expired - Fee Related CN101235379B (en)

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CN105255861B (en) * 2015-11-23 2019-01-08 广西大学 Directly extract the method and kit of 3 kinds of DNA of pathogenic in milk
CN108424908A (en) * 2018-05-09 2018-08-21 上海普洛麦格生物产品有限公司 A kind of nucleic acid releasing agent and hepatitis B virus nucleic acid method for releasing
CN114657174B (en) * 2022-03-29 2023-07-25 湖南科技学院 Kit for extracting bacterial plasmid by alkaline lysis method and method thereof

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CN1226600A (en) * 1999-01-29 1999-08-25 朱明华 Reagent box for extracting DNA fast and use thereof

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Publication number Priority date Publication date Assignee Title
CN1226600A (en) * 1999-01-29 1999-08-25 朱明华 Reagent box for extracting DNA fast and use thereof

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Title
姚伟 等.质粒DNA小量提取法的改进.应用与环境生物学报11 6.2005,11(6),776-778.
姚伟 等.质粒DNA小量提取法的改进.应用与环境生物学报11 6.2005,11(6),776-778. *
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王友如.大肠杆菌质粒DNA提取方法的优化.生物技术16 6.2006,16(6),42-44. *

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