CN103898095A - Soil nematode community genome extraction method - Google Patents
Soil nematode community genome extraction method Download PDFInfo
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- CN103898095A CN103898095A CN201410104236.XA CN201410104236A CN103898095A CN 103898095 A CN103898095 A CN 103898095A CN 201410104236 A CN201410104236 A CN 201410104236A CN 103898095 A CN103898095 A CN 103898095A
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Abstract
The invention relates to a soil nematode community genome extraction method. The method comprises the following specific steps: refrigerating an extracted soil nematode suspension at the temperature of 20 DEG C below zero for 10-20 minutes, defrosting at the room temperature, centrifuging the nematode suspension, and removing supernatant; adding a DNA (Deoxyribose Nucleic Acid) extracting solution and protease K into a centrifugal tube with nematode sediment at the temperature of 37 DEG C; adding a mixed solution of isometric phenol, chloroform and isoamylol into the heat-preserved and digested nematode solution in an equal volume, and centrifuging; centrifuging the nematode solution at the temperature of 4 DEG C, and centrifuging at the speed of 12,000 revolutions per minute for 10-12 minutes; sucking the supernatant into a new tube, adding sodium acetate in an amount which is 1/5 the volume of the supernatant and absolute ethyl alcohol in an amount which is 2 times the volume of the supernatant, and mixing; adding 70 percent ethanol into the centrifugal tube with sediment, washing DNA for 1-2 times, and centrifuging; naturally drying the obtained soil nematode DNA in the air, dissolving into sterilized double distilled water, and refrigerating at the temperature of 20 DEG C below zero for storage in order to reduce the interference with subsequent experiments. In the method, nematode cells are cracked by combining a physical method, a chemical method and an enzymic method, so that the DNA extraction effect of a nematode community sample genome is improved.
Description
Technical field
The present invention relates to gene and extract field, relate in particular to a kind of Soil nematode community Extraction Methods of Genome.
Background technology
Soil nematodes is the important metazoan in plant rhizosphere soil biotic district, huge in occurring in nature quantity, of a great variety, and participate in material cycle and the flow of energy of soil ecosystem directly, to organic decomposition, Nutrient Transformation and transmission ofenergy play crucial effect, are the important component parts of soil ecosystem.Soil nematodes viability, fecundity and adaptive capacity to environment are strong, and to environmental change sensitivity, its group's composition can reflect physics and chemistry and the biological character of soil, and therefore soil nematodes becomes the important indicator organism of evaluating soil health.
Because the nematode bodily form is less, morphological feature is subject to various factors, makes a variation larger, identifies these line insect types from morphology, needs abundant classification to identify knowledge and experience.Molecular biology method has become highly effective supplementary means, and its basis be simply, efficiently quick, the stable nematode DNA extraction of carrying out.Extract efficiently nematode communities DNA, make it to represent whole group, and then can carry out species diversity, functional diversity analysis.In recent years, alkaline lysis, Proteinase K method, test kit method etc. are carried out nematode DNA extraction, but have the problems such as experimental period is long, agents useful for same is many, price, unstable result.
In view of above-mentioned defect, creator of the present invention has obtained this creation finally through long research and practice.
Summary of the invention
The object of the present invention is to provide a kind of Soil nematode community Extraction Methods of Genome, in order to overcome above-mentioned technological deficiency.
For achieving the above object, the invention provides a kind of Soil nematode community Extraction Methods of Genome, this detailed process is:
Step a thawed the soil nematodes suspension of extraction after-20 ℃ of freezing 10-20 minutes under room temperature, by centrifugal nematode suspension, and reject supernatant liquor;
Step b, adds DNA extraction liquid and Proteinase K to leaving in the centrifuge tube of nematode precipitation, and 37 ℃, 150rp/min concussion is incubated digestion for 1-2 hour;
Step c, to by adding the mixed solution of isopyknic phenol, chloroform and primary isoamyl alcohol in the nematode solution of insulation digestion, centrifugal, supernatant liquor is transferred in another new pipe; Described nematode solution 4 ℃ centrifugal, with the centrifugal 10-12 minute of speed of 12000rp/m;
Steps d, draws supernatant liquor and adds the sodium-acetate of 1/5 times of volume and 2 times of volume dehydrated alcohols to mix in new Guan Zhongzai, leave standstill 2 hours in 4 ℃, then with 4 ℃, centrifugal 20 minutes of 14000rp/min, is soil nematode DNA, after mixed solution is centrifugal, and reject supernatant liquor;
Step e, adds 70% washing with alcohol DNA1-2 time to leaving in the centrifuge tube of precipitation, centrifugal, reject supernatant liquor;
Step f after naturally drying, adds aseptic double-distilled water dissolving DNA, freezing preservation in centrifuge tube; After gained soil nematode DNA natural air drying, be dissolved in sterilizing distilled water in-20 ℃ of freezing preservations, reduced the interference to subsequent experimental.
Further, in above-mentioned steps a, described nematode suspension is the nematode that sterilized water is preserved; Described centrifugal under room temperature, with the centrifugal 5min of speed of 8000rp/min.
Further, in above-mentioned steps a, pedotheque is added water filtration by being separated into of described Soil nematode community, pass through respectively one deck nematode filter paper and two-layer gauze, the nematode suspension then filtration being obtained leaves standstill about 30min, discards supernatant liquid, obtains concentrated nematode suspension.
Further, in above-mentioned steps a, described freeze thawing treatment: the soil nematodes suspension of extraction was thawed after-20 ℃ of freezing 10-20 minutes under room temperature.
Further, in above-mentioned steps b, described DNA extraction liquid is preserved under room temperature, is made into pH8.0 by 10mMTris-HCI, 1mMEDTA, 0.1% (V/V) SDS.
Further, in above-mentioned steps c, in the mixed solution of described phenol, chloroform and primary isoamyl alcohol, the volume ratio of phenol, chloroform and primary isoamyl alcohol is 25: 24: 1.
Further, in above-mentioned steps d, the concentration of described sodium-acetate is 3M, and dehydrated alcohol is-20 ℃ of precoolings.
Further, in above-mentioned steps e, 70% washing with alcohol 1-2 time for gained soil nematode DNA.
Further, in above-mentioned steps f, after gained soil nematode DNA natural air drying, be dissolved in aseptic double-distilled water and at-20 ℃ and saved backup.
Beneficial effect of the present invention is compared with prior art: the present invention adopts physics, chemistry, the enzyme process method that combines to carry out cracking to elegans cell, has improved nematode communities sample gene group DNA extraction effect.The DNA sample extracting can meet take PCR as basic reaction, and can be applied to other DNA operative technique, for example denaturing gradient gel electrophoresis, clone etc.
Accompanying drawing explanation
Fig. 1 is that different areas, Shaanxi Province of the present invention Soil nematode community DNA is in the agarose gel electrophoresis detection figure of 1% concentration.
Embodiment
Below in conjunction with accompanying drawing, technical characterictic and the advantage with other above-mentioned to the present invention are described in more detail.
The detailed process that this genome extracts is:
Step a thawed the soil nematodes suspension of extraction after-20 ℃ of freezing 10-20 minutes under room temperature, by centrifugal nematode suspension, and reject supernatant liquor; In this step, described nematode suspension is the nematode that sterilized water is preserved; Described centrifugal under room temperature, with the centrifugal 5min of speed of 8000rp/min.
Pedotheque is added water filtration by being separated into of described Soil nematode community, passes through respectively one deck nematode filter paper and 2 layers of gauze, and the nematode suspension then filtration being obtained leaves standstill about 30min, discards supernatant liquid, obtains concentrated nematode suspension.
Step b, adds DNA extraction liquid and Proteinase K to leaving in the centrifuge tube of nematode precipitation, and 37 ℃, 150rp/min concussion is incubated digestion for 1-2 hour; Described DNA extraction liquid is preserved under room temperature, is made into pH8.0 by 10mMTris-HCI, 1mMEDTA, 0.1% (V/V) SDS.
Step c, to by adding the mixed solution of isopyknic phenol, chloroform and primary isoamyl alcohol in the nematode solution of insulation digestion, centrifugal, supernatant liquor is transferred in another new pipe; In the mixed solution of described phenol, chloroform and primary isoamyl alcohol, the volume ratio of phenol, chloroform and primary isoamyl alcohol is 25: 24: 1, described centrifugal in 4 ℃, with the centrifugal 10-12 minute of speed of 12000rp/m.
Steps d, draws supernatant liquor and adds the NaAc of 1/5 times of volume and 2 times of volume dehydrated alcohols to mix in new Guan Zhongzai, leave standstill 2 hours in 4 ℃, then with 4 ℃, centrifugal 20 minutes of 14000rp/min, is soil nematode DNA, after mixed solution is centrifugal, and reject supernatant liquor; The concentration of described sodium-acetate is 3M, and dehydrated alcohol is-20 ℃ of precoolings.
Step e, adds 70% washing with alcohol DNA1-2 time to leaving in the centrifuge tube of precipitation, centrifugal, reject supernatant liquor;
Step f after naturally drying, adds aseptic double-distilled water dissolving DNA, freezing preservation in centrifuge tube; After gained soil nematode DNA natural air drying, be dissolved in sterilizing distilled water in-20 ℃ of freezing preservations, reduced the interference to subsequent experimental.
Refer to shown in Fig. 1, it is that different areas, Shaanxi Province Soil nematode community DNA detects figure in the agarose gel electrophoresis of 1% concentration;
In figure, M: standard; YJ: Yijun County soil nematodes; HPS: Hao Ping temple soil nematodes; LN: Luonan County soil nematodes.
Now according to embodiment, the present invention will be described:
Embodiment mono-:
Mixing with soil nematode sample obtains: the glass funnel end that is 20cm at bore connects one section of rubber tubing, clamp with spring pinchcock in rubber tubing rear end, in funnel, place one deck wire netting, on it, place two-layer gauze, and put one deck nematode filter paper in the above, press from both sides funnel is fixed on iron stand with funnel, get 200g soil sample uniform spreading on filter paper, add water to submergence soil.Due to the hydrotaxis of nematode and the weight of self, enter in water through filter paper space successively, be gathered in the rubber tubing of funnel end.Nematode tripping device is placed under 20 ℃ of room temperatures and is separated.After 24h, slowly open spring pinchcock, the water in rubber tubing is collected in culture dish, leave standstill about 30min, under microscope, draw upper strata clear water with glue head dropper, only retain layer line worm suspension, then nematode suspension proceeded in 1.5ml centrifuge tube, be stored in-20 ℃ for subsequent use.
By the at room temperature centrifugal 5min of 8000rp/min of soil nematodes suspension extracting;
Draw supernatant liquid with rifle head, retain 300ul liquid below, add 100ulDNA Extraction buffer and 4ul Proteinase K (20ug/ul) at 37 ℃, to shake insulation digestion 1h; And add isopyknic phenol, chloroform, primary isoamyl alcohol mixed solution to mix gently in the postdigestive nematode solution of backward insulation, room temperature leaves standstill 10min, 4 ℃, the centrifugal 10min of 12000rp/min, supernatant is transferred in another new pipe, adds the NaAc of 1/5 times of volume and the dehydrated alcohol of 2 times of volumes to mix, mixture is placed 2h in 4 ℃, the centrifugal 20min of 14000rp/min, abandons supernatant, and precipitation is soil nematodes genomic dna.Describedly be deposited in natural air drying under room temperature, add 30ul sterilizing distilled water, save backup at-20 ℃.
Embodiment bis-:
Mixing with soil nematode sample is retrieved as modified Baermann funnel method, after filter paper and filtered through gauze, gets rubber tubing bottom water sample, leaves standstill, and collects nematode suspension.
By the at room temperature centrifugal 5min of 8000rp/min of soil nematodes suspension extracting, with rifle head absorption supernatant liquid, retain 200ul liquid below, add 100ulDNA Extraction buffer and 4ul Proteinase K (20ug/ul) at 37 ℃, to shake insulation digestion 2h; And add isopyknic phenol, chloroform, primary isoamyl alcohol mixed solution to mix gently in the postdigestive nematode solution of backward insulation, room temperature leaves standstill 10min, 4 ℃, the centrifugal 10min of 12000rp/min, supernatant is transferred in another new pipe, adds the NaAc of 1/10 times of volume and the dehydrated alcohol of 2 times of volumes to mix, mixture is placed 2h in 4 ℃, the centrifugal 20min of 14000rp/min, abandons supernatant, and precipitation is soil nematodes genomic dna.Described precipitation with after 70% washing with alcohol under room temperature natural air drying, add 50ul sterilizing distilled water, save backup at-20 ℃.
Embodiment tri-:
Mixing with soil nematode sample is retrieved as modified Baermann funnel method, after filter paper and filtered through gauze, gets rubber tubing bottom water sample, leaves standstill, and collects nematode suspension and saves backup at-20 ℃.
The centrifugal 5min of 8000rp/min at room temperature after the soil nematodes suspension room temperature of extraction is dissolved, with rifle head absorption supernatant liquid, retain 200ul liquid below, add 100ulDNA Extraction buffer and 4ul Proteinase K (20ug/ul) at 37 ℃, to shake insulation digestion 1.5h; And add isopyknic phenol, chloroform, primary isoamyl alcohol mixed solution to mix gently in the postdigestive nematode solution of backward insulation, room temperature leaves standstill 10min, 4 ℃, the centrifugal 10min of 12000rp/min, supernatant is transferred in another new pipe, adds the NaAc of 1/5 times of volume and the dehydrated alcohol of 2 times of volumes to mix, mixture is placed 2h in 4 ℃, the centrifugal 20min of 14000rp/min, abandons supernatant, and precipitation is soil nematodes genomic dna.Described precipitation with after 70% washing with alcohol under room temperature natural air drying, add 40ul sterilizing distilled water, save backup at-20 ℃.
The foregoing is only preferred embodiment of the present invention, is only illustrative for invention, and nonrestrictive.Those skilled in the art is understood, and in the spirit and scope that limit, can carry out many changes to it in invention claim, revise, and even equivalence, but all will fall within the scope of protection of the present invention.
Claims (9)
1. a Soil nematode community Extraction Methods of Genome, is characterized in that, this detailed process is:
Step a thawed the soil nematodes suspension of extraction after-20 ℃ of freezing 10-20 minutes under room temperature, by centrifugal nematode suspension, and reject supernatant liquor;
Step b, adds DNA extraction liquid and Proteinase K to leaving in the centrifuge tube of nematode precipitation, and 37 ℃, 150rp/min concussion is incubated digestion for 1-2 hour;
Step c, to by adding the mixed solution of isopyknic phenol, chloroform and primary isoamyl alcohol in the nematode solution of insulation digestion, centrifugal, supernatant liquor is transferred in another new pipe; Described nematode solution 4 ℃ centrifugal, with the centrifugal 10-12 minute of speed of 12000rp/m;
Steps d, draws supernatant liquor and adds the sodium-acetate of 1/5 times of volume and 2 times of volume dehydrated alcohols to mix in new Guan Zhongzai, leaves standstill 2 hours in 4 ℃, then with 4 ℃, centrifugal 20 minutes of 14000rp/min, reject supernatant liquor, precipitation is soil nematode DNA;
Step e, adds 70% washing with alcohol DNA1-2 time to leaving in the centrifuge tube of precipitation, centrifugal, reject supernatant liquor;
Step f after naturally drying, adds aseptic double-distilled water dissolving DNA, freezing preservation in centrifuge tube; After gained soil nematode DNA natural air drying, be dissolved in sterilizing distilled water in-20 ℃ of freezing preservations, reduced the interference to subsequent experimental.
2. Soil nematode community Extraction Methods of Genome according to claim 1, is characterized in that, in above-mentioned steps a, described nematode suspension is the nematode that sterilized water is preserved; Described centrifugal under room temperature, with the centrifugal 5min of speed of 8000rp/min.
3. Soil nematode community Extraction Methods of Genome according to claim 2, it is characterized in that, in above-mentioned steps a, pedotheque is added water filtration by being separated into of described Soil nematode community, pass through respectively one deck nematode filter paper and two-layer gauze, the nematode suspension then filtration being obtained leaves standstill about 30min, discards supernatant liquid, obtains concentrated nematode suspension.
4. Soil nematode community Extraction Methods of Genome according to claim 3, is characterized in that, in above-mentioned steps a, and described freeze thawing treatment: the soil nematodes suspension of extraction was thawed after-20 ℃ of freezing 10-20 minutes under room temperature.
5. Soil nematode community Extraction Methods of Genome according to claim 4, it is characterized in that, in above-mentioned steps b, described DNA extraction liquid is preserved under room temperature, be made into pH8.0 by 10mMTris-HCI, 1mMEDTA, 0.1% (V/V) SDS.
6. Soil nematode community Extraction Methods of Genome according to claim 1 or 5, is characterized in that, in above-mentioned steps c, in the mixed solution of described phenol, chloroform and primary isoamyl alcohol, the volume ratio of phenol, chloroform and primary isoamyl alcohol is 25: 24: 1.
7. Soil nematode community Extraction Methods of Genome according to claim 1 or 5, is characterized in that, in above-mentioned steps d, the concentration of described sodium-acetate is 3M, and dehydrated alcohol is-20 ℃ of precoolings.
8. Soil nematode community Extraction Methods of Genome according to claim 1 or 5, is characterized in that, 70% washing with alcohol 1-2 time for gained soil nematode DNA.
9. Soil nematode community Extraction Methods of Genome according to claim 1 or 5, is characterized in that, after gained soil nematode DNA natural air drying, is dissolved in aseptic double-distilled water and at-20 ℃ and saves backup.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106718181A (en) * | 2017-02-10 | 2017-05-31 | 北京农学院 | A kind of method of plant identification to root knot nematode resistance |
| CN108866047A (en) * | 2018-08-14 | 2018-11-23 | 南京林业大学 | A kind of genome DNA extracting method based on multigelation mode |
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| CN101580830A (en) * | 2008-05-15 | 2009-11-18 | 中国科学院沈阳应用生态研究所 | Soil nematode DNA extraction method |
| CN102703582A (en) * | 2012-03-19 | 2012-10-03 | 华南农业大学 | Method for direct rapid detection and identification of banana-root nematode in soil or other media |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101580830A (en) * | 2008-05-15 | 2009-11-18 | 中国科学院沈阳应用生态研究所 | Soil nematode DNA extraction method |
| CN102703582A (en) * | 2012-03-19 | 2012-10-03 | 华南农业大学 | Method for direct rapid detection and identification of banana-root nematode in soil or other media |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106718181A (en) * | 2017-02-10 | 2017-05-31 | 北京农学院 | A kind of method of plant identification to root knot nematode resistance |
| CN106718181B (en) * | 2017-02-10 | 2020-05-26 | 北京农学院 | Method for identifying resistance of plants to root-knot nematodes |
| CN108866047A (en) * | 2018-08-14 | 2018-11-23 | 南京林业大学 | A kind of genome DNA extracting method based on multigelation mode |
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