CN102220309A - Method for extracting DNA (deoxyribonucleic acid) of active sludge in anaerobic reactor - Google Patents
Method for extracting DNA (deoxyribonucleic acid) of active sludge in anaerobic reactor Download PDFInfo
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Abstract
The invention discloses a method for extracting DNA (deoxyribonucleic acid) of sludge in a sewage treatment reactor and particularly relates to a method for extracting DNA of sludge in an anaerobic reactor. DNA extraction is the basis for technical research of molecular biology, and the DNA extraction quality has great influence on subsequent research, i.e., high-quality DNA can provide accurate information for research of molecular biology; and the existing method for extracting DNA of anaerobic sludge generally has the defects of large extraction difficulty, many impurities and low yield, and restriction to research of people on anaerobic sludge to some degree. The invention provides the effective method for extracting the DNA of sludge in the anaerobic reactor, thus the defects of the existing method can be solved. The DNA extracted by the method disclosed by the invention has the characteristics of large yield and high purity, and is easy to extract.
Description
Technical field
The present invention relates to the extracting method of activated sludge DNA in the wastewater treatment biochemical reactor, specifically is the extracting method of activated sludge DNA in a kind of anaerobic reactor.
Background technology
The anaerobic biological treatment reactor is the hot topic of present sewage treatment area research, the microorganism of research anaerobic biological treatment reactor has the important meaning for the reaction mechanism that discloses anaerobic treatment, traditional microorganism separates pure culture technigne and is difficult to comprehensively objectively research anaerobion wherein, educable microorganism is less than 1% of occurring in nature microbial count in the laboratory at present, anaerobion still less and also has much because of cultivating not the microorganism by people were familiar with.The introducing of a Protocols in Molecular Biology difficult problem has hereto had radical solving method, makes people break away from dependence to culture technique, from the characteristic of molecule angle research microorganism, has promoted the development of anaerobism technology greatly.
Extracting high-quality total DNA from environmental sample, is the precondition of carrying out PCR, DGGE etc., and multifarious analysis plays crucial effects to the quality of DNA extraction for flora.The DNA that extracts from environmental sample requires be able to represent the practical situation of genetic diversity in this sample, PCR, DGGE etc. have very high requirement to DNA, and it is crucial therefore selecting suitable DNA extraction method targetedly and extract high-quality DNA.
Because the anaerobion in the anaerobic reactor mud is comparatively complicated, and be difficult to broken wall, often yield is less for common DNA extraction method, and owing to contained humic acids, protein and glucide in the anaerobic reactor mud are more, therefore be difficult to extract the higher DNA of purity usually, and these materials can produce inhibition and interfering influence to subsequent P CR reaction, and then influence DGGE, connection and the conversion etc. of back.
Summary of the invention
There is the shortcoming that the extraction difficulty is big, impurity is many, yield poorly in the extracting method that the present invention is directed to activated sludge DNA in the existing anaerobic reactor, and the extracting method of activated sludge DNA in a kind of anaerobic reactor is provided.The present invention extracts the method for anaerobic reactor mud DNA and carries out as follows:
(1) gets about 30ml sewage sample, under 4 ℃, with 9000 rev/mins rotating speed centrifugal 5 minutes, remove liquid, collecting precipitation;
(2) TENP damping fluid and 3 the sterilization granulated glass spherees that add 5 times of precipitation volumes fully shook 5 minutes, under 4 ℃, with 9000 rev/mins rotating speed centrifugal 5 minutes, removed liquid, collecting precipitation (repeating this step comparatively clarifies up to supernatant);
(3) add the PBS damping fluid of 5 times of precipitation volumes, fully concussion under 4 ℃, with 9000 rev/mins rotating speed centrifugal 5 minutes, is removed liquid, collecting precipitation (this step repeats 2 times);
(4) the PBS damping fluid of 5 times of precipitation volumes of adding suspends;
(5) collect the good mud sample of 1g pre-treatment, add 200 μ L and extract damping fluid, add 2 granulated glass spherees, fully shook 5 minutes, sample is fully suspended;
(6) suspension is placed on-25 ℃ of refrigerator and cooled and freezes and put into 65～70 ℃ of water-baths heating 30 minutes after 30 minutes, rocked once, make the abundant cracking of cell (this step repeats 3 times) every 10 minutes;
(7) add 4 μ L N,O-Diacetylmuramidases (50mg/mL), room temperature was placed 5 minutes, placed 94 ℃ of water-baths to place then 1 minute;
(8) add 400 μ L SDS damping fluids, 1 μ L Proteinase K (20mg/mL), 1.5 μ L RNA lytic enzymes (20mg/ml) mix to be placed in 37 ℃ of water-baths and placed 1 hour;
(9) add and the saturated phenol of mixed solution equal-volume Tris-, mixing, at room temperature, 12000 rev/mins centrifugal 5 minutes, supernatant liquor is changed in another centrifuge tube;
(10) add and supernatant liquor equal-volume mixed solution, mixed solution consists of phenol/chloroform/primary isoamyl alcohol, and phenol/chloroform/primary isoamyl alcohol volume ratio is 25: 24: 1, mixing, at room temperature, 12000 rev/mins centrifugal 5 minutes, draw supernatant liquor to another centrifuge tube;
(11) add the Virahol mixing of 0.6 times of supernatant liquor volume, room temperature was placed 2 hours, at room temperature, 12000 rev/mins centrifugal 10 minutes, abandon supernatant liquor;
(12) precipitation is with 70% washing with alcohol 3 times, adding sterile purified water dissolution precipitation after the drying at room temperature, and gained is DNA.
The major function of PVP (polyvinylpyrrolidone) in the extracting method step (2) of anaerobic reactor mud DNA of the present invention in the used TENP damping fluid is to remove soil ulmin, because the humus content in the anaerobic reactor is big, humic acids contained in the soil ulmin can play restraining effect to the PCR reaction of back, the TENP damping fluid can effectively be removed soil ulmin, improves the purity of DNA.
In the extracting method step (6) of activated sludge DNA suspension being placed on-25 ℃ of refrigerator and cooled in the anaerobic reactor of the present invention freezes and puts into 65～70 ℃ of water-baths heating 30 minutes after 30 minutes, rocked once every 10 minutes, repeat this step 3 time, the principle of multigelation is to form salt concn that ice pellets makes remaining cell liquid in owing to cell to increase and cause cytoclasis in the time of-25 ℃.
Positively effect of the present invention is, by adding the use of TENP damping fluid and granulated glass sphere to anaerobic sludge The pretreatment and multigelation method, the DNA extraction efficient of anaerobic sludge is improved greatly, big, the easy extraction of the DNA output that utilization present method is extracted, purity height.In addition, present method is workable, and TENP damping fluid compound method is simple, and granulated glass sphere low price, refrigerator and water-bath all are the laboratory common instruments, do not need specific apparatus, and experimental cost is lower.
Description of drawings
Fig. 1 is the agarose gel electrophoresis comparison diagram of activated sludge DNA in activated sludge DNA and embodiment two extract in the anaerobic reactor that present method extracts the anaerobic reactor, and Fig. 2 is deformation gradient gel electrophoresis (DGGE) comparison diagram of activated sludge DNA in the anaerobic reactor of activated sludge DNA in the anaerobic reactor of present method extraction and embodiment two extractions.
Embodiment
The present invention is further elaborated below in conjunction with concrete test, but embodiments of the present invention are not limited thereto.
Embodiment one:
(1) get respectively in the anaerobic reactor before in the about 30ml of sewage sample in back 3 lattice chambers, number 1,2 and 3 respectively, under 4 ℃, with 9000 rev/mins rotating speed centrifugal 5 minutes, remove liquid, collecting precipitation;
(2) TENP damping fluid and 3 the sterilization granulated glass spherees that add 5 times of precipitation volumes fully shook 5 minutes, under 4 ℃, with 9000 rev/mins rotating speed centrifugal 5 minutes, removed liquid, collecting precipitation; (repeating this step comparatively clarifies up to supernatant)
(3) add the PBS damping fluid of 5 times of precipitation volumes, fully concussion under 4 ℃, with 9000 rev/mins rotating speed centrifugal 5 minutes, is removed liquid, collecting precipitation (this step repeats 2 times);
(4) the PBS damping fluid of 5 times of precipitation volumes of adding suspends;
(5) collect the good mud sample of 1g pre-treatment, add 200 μ l and extract damping fluid, add 2 granulated glass spherees, fully shook 5 minutes, sample is fully suspended;
(6) suspension is placed on-25 ℃ of refrigerator and cooled and freezes and put into 65～70 ℃ of water-baths heating 30 minutes after 30 minutes, rocked once, make the abundant cracking of cell (this step repeats 3 times) every 10 minutes;
(7) add 4 μ L N,O-Diacetylmuramidases (50mg/mL), room temperature was placed 5 minutes, placed 94 ℃ of water-baths to place then 1 minute;
(8) add 400 μ L SDS damping fluids, 1 μ L Proteinase K (20mg/mL), 1.5 μ L RNA lytic enzymes (20mg/ml) mix to be placed in 37 ℃ of water-baths and placed 1 hour;
(9) add and the saturated phenol of mixed solution equal-volume Tri s-, mixing, at room temperature, 12000 rev/mins centrifugal 5 minutes, supernatant liquor is changed in another centrifuge tube;
(10) add and supernatant liquor equal-volume mixed solution, mixed solution is formed phenol: chloroform: primary isoamyl alcohol volume ratio 25: 24: 1, mixing, at room temperature, 12000 rev/mins centrifugal 5 minutes, draw supernatant liquor to another centrifuge tube;
(11) add the Virahol mixing of 0.6 times of supernatant liquor volume, room temperature was placed 2 hours, at room temperature, 12000 rev/mins centrifugal 10 minutes, abandon supernatant liquor;
(12) precipitation is with 70% washing with alcohol 3 times, adding sterile purified water dissolution precipitation after the drying at room temperature, and gained is DNA.
Embodiment two:
(1) get respectively in the anaerobic reactor before in the about 30ml of sewage sample in back 3 lattice chambers, number 1 ', 2 ' and 3 ' respectively, under 4 ℃, with 9000 rev/mins rotating speed centrifugal 5 minutes, remove liquid, collecting precipitation;
(2) add the PBS damping fluid of 5 times of precipitation volumes, fully concussion under 4 ℃, with 9000 rev/mins rotating speed centrifugal 5 minutes, is removed liquid, collecting precipitation; (this step repeats 2 times)
(3) the PBS damping fluid of 5 times of precipitation volumes of adding suspends.
(4) collect the good mud sample of 1g pre-treatment, add 200 μ l and extract damping fluid, add 2 granulated glass spherees, fully shook 5 minutes, sample is fully suspended;
(5) add 4 μ L N,O-Diacetylmuramidases (50mg/mL), room temperature was placed 5 minutes, placed 94 ℃ of water-baths to place then 1 minute;
(6) add 400 μ L SDS damping fluids, 1 μ L Proteinase K (20mg/mL), 1.5 μ L RNA lytic enzymes (20mg/ml) mix to be placed in 37 ℃ of water-baths and placed 1 hour;
(7) add and the saturated phenol of mixed solution equal-volume Tris-, mixing, at room temperature, 12000 rev/mins centrifugal 5 minutes, supernatant liquor is changed in another centrifuge tube;
(8) add and supernatant liquor equal-volume mixed solution, mixed solution is formed phenol/chloroform/primary isoamyl alcohol, and volume ratio is 25: 24: 1, mixing, at room temperature, 12000 rev/mins centrifugal 5 minutes, draw supernatant liquor to another centrifuge tube;
(9) add the Virahol mixing of 0.6 times of supernatant liquor volume, room temperature was placed 2 hours, at room temperature, 12000 rev/mins centrifugal 10 minutes, abandon supernatant liquor;
(10) precipitation is with 70% washing with alcohol 3 times, adding sterile purified water dissolution precipitation after the drying at room temperature, and gained is DNA.
As can be seen from Figure 1, the DNA band ( band 1,2 and 3) that present embodiment is extracted is clear bright, does not have obviously assorted band, and with the DNA band (1 ', 2 ' and 3 ') of existing method extraction hardly as seen, as seen, the existing method of the DNA rate ratio of present embodiment extraction is significantly improved.
As can be seen from Figure 2, the DNA that extracts with present embodiment is that the DGGE band done of masterplate is high-visible, band quantity is many, diversity is good, can embody the microorganism richness in the anaerobic reactor preferably, the DNA that extracts with existing method is that the DGGE band done of masterplate is unintelligible, and band quantity is few, diversity is poor, can not embody the microorganism richness in the anaerobic reactor preferably.
Claims (4)
1. the extracting method of a sewage disposal anaerobic reactor activated sludge DNA is characterized in that the method for extracting the anaerobic reactor activated sludge DNA carries out as follows:
(1) gets about 30ml sewage sample, under 4 ℃, with 9000 rev/mins rotating speed centrifugal 5 minutes, remove liquid, collecting precipitation;
(2) TENP damping fluid and 3 the sterilization granulated glass spherees that add 5 times of precipitation volumes fully shook 5 minutes, under 4 ℃, and with 9000 rev/mins rotating speed centrifugal 5 minutes, remove liquid, collecting precipitation repeats this step and comparatively clarifies up to supernatant;
(3) add the PBS damping fluid of 5 times of precipitation volumes, fully concussion, under 4 ℃, with 9000 rev/mins rotating speed centrifugal 5 minutes, remove liquid, collecting precipitation, this step repeats 2 times;
(4) the PBS damping fluid of 5 times of precipitation volumes of adding suspends;
(5) collect the good mud sample of 1g pre-treatment, add 200 μ l and extract damping fluid, add 2 granulated glass spherees, fully shook 5 minutes, sample is fully suspended;
(6) suspension is placed on-25 ℃ of refrigerator and cooled and freezes and put into 65～70 ℃ of water-baths heating 30 minutes after 30 minutes, rocked once every 10 minutes, make the abundant cracking of cell, this step repeats 3 times;
(7) adding 4 μ L concentration is the N,O-Diacetylmuramidase of 50mg/mL, and room temperature was placed 5 minutes, places 94 ℃ of water-baths to place then 1 minute;
(8) add 400 μ L SDS damping fluids, 1 μ L concentration is the Proteinase K of 20mg/mL, and 1.5 μ L concentration are the RNA lytic enzyme of 20mg/mL, mixes to be placed in 37 ℃ of water-baths and places 1 hour;
(9) add and the saturated phenol of mixed solution equal-volume Tris-, mixing, at room temperature, 12000 rev/mins centrifugal 5 minutes, supernatant liquor is changed in another centrifuge tube;
(10) add and supernatant liquor equal-volume mixed solution, mixed solution consists of phenol/chloroform/primary isoamyl alcohol, and phenol/chloroform/primary isoamyl alcohol volume ratio is 25: 24: 1, mixing, at room temperature, 12000 rev/mins centrifugal 5 minutes, draw supernatant liquor to another centrifuge tube;
(11) add the Virahol mixing of 0.6 times of supernatant liquor volume, room temperature was placed 2 hours, at room temperature, 12000 rev/mins centrifugal 10 minutes, abandon supernatant liquor;
(12) precipitation is with 70% washing with alcohol 3 times, adding sterile purified water dissolution precipitation after the drying at room temperature, and gained is DNA.
2. press the extracting method of activated sludge DNA in the described a kind of anaerobic reactor of claim 1, it is characterized in that: TENP damping fluid and 3 sterilization granulated glass spherees of adding 5 times of precipitation volumes in the step (2) fully shook 5 minutes, repeated this step and comparatively clarified up to supernatant.
3. press the extracting method of activated sludge DNA in the described a kind of anaerobic reactor of claim 1, it is characterized in that: in the step (2) in the used TENP damping fluid component concentrations be respectively: 50mM Tris, 20mM EDTA, 100mM NaCl, 0.01g/ml PVP, the pH value of TENP damping fluid is 10.
4. press the extracting method of the described a kind of anaerobic reactor activated sludge DNA of claim 1, it is characterized in that: in the step (6) suspension is placed on-25 ℃ of refrigerator and cooled and freezes and put into 65～70 ℃ of water-baths heating 30 minutes after 30 minutes, rocked once every 10 minutes, repeat this step 3 time.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102409041A (en) * | 2011-12-08 | 2012-04-11 | 华东师范大学 | Extraction method of total genome DNA from microbes |
| CN102703436A (en) * | 2012-06-15 | 2012-10-03 | 赣南师范学院 | Method for extracting DNA of citrus canker by water bath precipitation |
| CN103215254A (en) * | 2013-04-26 | 2013-07-24 | 东华大学 | Method for extracting activated sludge sample DNA (Deoxyribonucleic Acid) in SBR (Styrene Butadiene Rubber) |
| CN103710337A (en) * | 2013-12-24 | 2014-04-09 | 齐鲁工业大学 | Method for efficiently extracting total DNA (deoxyribonucleic acid) of anaerobic granular sludge microorganisms |
| CN108034653A (en) * | 2018-04-07 | 2018-05-15 | 海南大学 | A kind of bacterium method for extracting total RNA of efficient stable |
| CN110672485A (en) * | 2019-09-06 | 2020-01-10 | 浙江大学 | Method for accurately measuring surface hydrophobicity of activated sludge by adsorbing dye |
| CN111065451A (en) * | 2017-07-18 | 2020-04-24 | 海岸线生物有限责任公司 | Semi-dry bead milling method for microbial lysis and device for carrying out same |
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| CN101696410A (en) * | 2009-10-26 | 2010-04-21 | 河海大学 | DNA extraction method suitable for structural analysis of microbial community in sediment |
| CN101935643A (en) * | 2010-08-19 | 2011-01-05 | 中国水产科学研究院黄海水产研究所 | Method for efficiently extracting biological total DNA from Apostichopus japonicus intestinal content and environmental substrate sludge |
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2011
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| CN101696410A (en) * | 2009-10-26 | 2010-04-21 | 河海大学 | DNA extraction method suitable for structural analysis of microbial community in sediment |
| CN101935643A (en) * | 2010-08-19 | 2011-01-05 | 中国水产科学研究院黄海水产研究所 | Method for efficiently extracting biological total DNA from Apostichopus japonicus intestinal content and environmental substrate sludge |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102409041A (en) * | 2011-12-08 | 2012-04-11 | 华东师范大学 | Extraction method of total genome DNA from microbes |
| CN102703436A (en) * | 2012-06-15 | 2012-10-03 | 赣南师范学院 | Method for extracting DNA of citrus canker by water bath precipitation |
| CN103215254A (en) * | 2013-04-26 | 2013-07-24 | 东华大学 | Method for extracting activated sludge sample DNA (Deoxyribonucleic Acid) in SBR (Styrene Butadiene Rubber) |
| CN103710337A (en) * | 2013-12-24 | 2014-04-09 | 齐鲁工业大学 | Method for efficiently extracting total DNA (deoxyribonucleic acid) of anaerobic granular sludge microorganisms |
| CN103710337B (en) * | 2013-12-24 | 2015-12-02 | 齐鲁工业大学 | A kind of method of high efficiency extraction anaerobic grain sludge microorganism total DNA |
| CN111065451A (en) * | 2017-07-18 | 2020-04-24 | 海岸线生物有限责任公司 | Semi-dry bead milling method for microbial lysis and device for carrying out same |
| CN108034653A (en) * | 2018-04-07 | 2018-05-15 | 海南大学 | A kind of bacterium method for extracting total RNA of efficient stable |
| CN110672485A (en) * | 2019-09-06 | 2020-01-10 | 浙江大学 | Method for accurately measuring surface hydrophobicity of activated sludge by adsorbing dye |
| CN110672485B (en) * | 2019-09-06 | 2020-07-28 | 浙江大学 | Method for accurately measuring surface hydrophobicity of activated sludge by adsorbing dye |
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Application publication date: 20111019 |