CN106497913A - A kind of method for extracting soil microorganism STb gene and its application - Google Patents
A kind of method for extracting soil microorganism STb gene and its application Download PDFInfo
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Abstract
The present invention relates to microorganism field, disclose a kind of method for extracting soil microorganism STb gene and its application in soil micro-ecosystem analysis, methods described includes the process of soil microorganism cell breakage, DNA purification process and DNA dissolution process, wherein, pretreatment was carried out to soil before the process of soil microorganism cell breakage, and the pretreatment includes washing soil with Polymeric sodium metaphosphate. solution and washs soil with isopropanol.The method of the extraction soil microorganism STb gene of the present invention, can efficiently extract the STb gene of soil microorganism, and Product yields are big, purity is high, disclosure satisfy that all kinds of subsequent operation requirements.
Description
Technical field
The present invention relates to microorganism field, in particular it relates to a kind of extraction soil microorganism STb gene
Method and its application in soil micro-ecosystem analysis.
Background technology
Traditional soil micro-ecosystem analysis is to carry out separation and Culture by the microorganism in soil mostly, sees
Examine its growth traits and Physiology and biochemistry property.However, due to the micro- life for having up to 99%-99.9% in soil
Thing is difficult to separation and Culture, and this traditional method has compared with big limitation.Therefore there has been proposed microorganism point
Sub- ecology technology, directly studies the Tiny ecosystem of soil using Molecular tools.And Molecular Ecology of Microbiology
A step for being applied to the key of soil micro-ecosystem research is exactly extraction microorganism total DNA from soil.
The extraction of soil microorganism STb gene, a main difficult problem are that the possible adsorbed close of soil particle is micro-
Biomass or DNA, cause to extract difficulty；Contain a large amount of humic acid, phenols, weight in soil simultaneously
The impurity such as metal ion, directly affect the operation such as follow-up PCR, enzyme action.General soil microbial total
The extraction of DNA includes Physical (such as freeze thawing, grinding, ultrasonication etc.), and chemical method (such as uses table
Face activating agent etc.) and bioanalysises (such as using lysozyme, protease etc.).Various methods are respectively provided with difference
Merits and demerits, be incorporated into the research direction for being current main flow.
At present, the methods such as high-temperature cracking method, SDS-CTAB cracking processs, enzymatic lysises method have been had been reported that
The extraction of soil microorganism STb gene is carried out, also has commercial kit to list.But these methods are obtained
The DNA output for arriving is less, and impurity removal rate can not meet demand.
Content of the invention
The invention aims to overcome prior art method extract the DNA output that obtains less,
A kind of relatively low defect of purity, there is provided method of extraction soil microorganism STb gene and its in the micro- life of soil
Application in state analysis.The method of the present invention can efficiently extract the STb gene of soil microorganism, and
Product yields are big, purity is high, disclosure satisfy that all kinds of subsequent operation requirements.
The present inventor has been surprisingly found that under study for action, when soil microorganism STb gene is extracted, leads to
Crossing to take before the process of soil microorganism cell breakage was included being washed with Polymeric sodium metaphosphate. solution to soil
Wash soil and the mode of the pretreatment of soil is washed with isopropanol, can significantly improve the DNA's that obtains
Yield and purity.
Therefore, to achieve these goals, in a first aspect, the invention provides a kind of extract the micro- life of soil
The method of thing STb gene, methods described include the process of soil microorganism cell breakage, DNA purification process
With DNA dissolution process, wherein, pre- place is carried out to soil before the process of soil microorganism cell breakage
Reason, the pretreatment include washing soil with Polymeric sodium metaphosphate. solution and wash soil with isopropanol.
Second aspect, the invention provides application of the said method in soil micro-ecosystem analysis.
The method of the extraction soil microorganism STb gene of the present invention, can efficiently extract from dissimilarity
The STb gene of the microorganism of matter soil, and Product yields are big, purity is high, disclosure satisfy that and exist including PCR
Interior all kinds of subsequent operations are required.
Other features and advantages of the present invention will be described in detail in subsequent specific embodiment part.
Specific embodiment
Hereinafter the specific embodiment of the present invention is described in detail.It should be appreciated that this place is retouched
The specific embodiment that states is merely to illustrate and explains the present invention, is not limited to the present invention.
In a first aspect, the invention provides a kind of method for extracting soil microorganism STb gene, the method
Including the process of soil microorganism cell breakage, DNA purification process and DNA dissolution process, wherein, exist
Pretreatment is carried out to soil before the process of soil microorganism cell breakage, and the pretreatment includes using Metaphosphoric acid
Sodium solution washs soil and washs soil with isopropanol.
In the inventive method, for washing soil with Polymeric sodium metaphosphate. solution in pretreatment and washed with isopropanol
The order of soil is not particularly required, then can be used again for first washing soil with Polymeric sodium metaphosphate. solution
Isopropanol washs soil, or and soil is first washed with isopropanol, is then washed with Polymeric sodium metaphosphate. solution again
Wash soil.In order to further improve the yield and purity of extracting the DNA for obtaining, under preferable case, in advance
First soil is washed with Polymeric sodium metaphosphate. solution in process, then wash soil with isopropanol again.
In the inventive method, when soil is washed with Polymeric sodium metaphosphate. solution, dense for Polymeric sodium metaphosphate. solution
There is no particular limitation with pH for degree, can be various concentration commonly used in the art and pH, under preferable case,
The concentration of Polymeric sodium metaphosphate. solution is 15-25g/L, and pH is 8-9.It will be understood by those skilled in the art that
In the inventive method, in Polymeric sodium metaphosphate. solution used, solute is only Polymeric sodium metaphosphate., does not include other compositions,
Such as PVP (polyvinylpyrrolidone).
In the inventive method, when soil is washed with Polymeric sodium metaphosphate. solution, for the use of Polymeric sodium metaphosphate. solution
There is no particular limitation for amount, as long as can be washed to soil using Polymeric sodium metaphosphate. solution, preferably
In the case of, relative to every gram of soil, the consumption of Polymeric sodium metaphosphate. solution is 2-6mL.
In the inventive method, when soil is washed with Polymeric sodium metaphosphate. solution, in order to further improve
The yield and purity of the DNA for arriving, under preferable case, the number of times for washing soil with Polymeric sodium metaphosphate. solution is
2-4.
In the inventive method, when soil being washed with Polymeric sodium metaphosphate. solution, under preferable case, use Metaphosphoric acid
The mode of sodium solution washing soil includes：Polymeric sodium metaphosphate. solution is added in soil, is centrifuged after stirring
Take precipitation.For the condition being centrifuged, there is no particular limitation, can be various centrifugation bars commonly used in the art
Part, for example, can be room temperature centrifugation 8-12min under 10000-13000rpm.
In the inventive method, when soil is washed with isopropanol, for the consumption of isopropanol does not have particularly
Limit, as long as can be washed to soil using isopropanol, under preferable case, relative to per gram
Soil, the consumption of isopropanol is 1-3mL.
In the inventive method, in order to further improve the yield and purity of extracting the DNA for obtaining, preferably
In the case of, the number of times for washing soil with isopropanol is 2-4.
In the inventive method, under preferable case, the mode for washing soil with isopropanol includes：In soil or
Add isopropanol, stirring is in precipitation (precipitation obtained after for example soil being washed through Polymeric sodium metaphosphate. solution)
Even rear centrifuging and taking precipitation.For the condition of centrifugation, there is no particular limitation, can be commonly used in the art each
Centrifugal condition is planted, for example, can be room temperature centrifugation 8-12min under 10000-13000rpm.
In the inventive method, in order to further improve the yield and purity of extracting the DNA for obtaining, preferably
In the case of, the pretreatment is also included with Crombach buffer solution soil.
In the inventive method, for being washed soil, used Crombach with Polymeric sodium metaphosphate. solution in pretreatment
Buffer solution soil and the order of soil is washed with isopropanol particularly do not require, can be with any suitable
Sequence washs soil.In order to further improve the yield and purity of extracting the DNA for obtaining, under preferable case,
Pretreatment includes washing soil with Polymeric sodium metaphosphate. solution, Crombach buffer and isopropanol successively.
In the inventive method, when with Crombach buffer solution soil, for Crombach delays
There is no particular limitation to rush pH, component and the concentration of liquid, can be respectively various pH commonly used in the art,
Component and concentration, under preferable case, Crombach buffer includes Tris-HCl and EDTA-Na2,
The concentration of Tris-HCl is 0.03-0.04mol/L, EDTA-Na2Concentration be 0.0005-0.0015mol/L,
The pH of Crombach buffer is 7.5-8.5.
In the inventive method, when with Crombach buffer solution soil, for Crombach delays
There is no particular limitation to rush the consumption of liquid, as long as can be washed to soil using Crombach buffer
Wash, under preferable case, it is preferable that relative to every gram of soil, the consumption of Crombach buffer
For 2-6mL.
In the inventive method, when with Crombach buffer solution soil, in order to further improve
The yield and purity of the DNA for obtaining, under preferable case, with Crombach buffer solution soil
Number of times be 2-4.
In the inventive method, when with Crombach buffer solution soil, under preferable case, use
The mode of Crombach buffer solution soil includes：Crombach bufferings are added in soil or precipitation
Liquid, centrifuging and taking precipitation after stirring.For the condition being centrifuged, there is no particular limitation, can be ability
The conventional various centrifugal conditions in domain, for example, can be room temperature centrifugation 8-12min under 10000-13000rpm.
In the inventive method, there is no particular limitation for the method processed for soil microorganism cell breakage,
Can be various soil microorganism cell breakage processing methods commonly used in the art, under preferable case, soil
Microbial cell breakage process includes：DNA extraction buffering is sequentially added in the sample that pretreatment is obtained
Liquid, lysozyme and E.C. 3.4.21.64 are processed.
In the inventive method, for component and its consumption, the consumption of lysozyme of DNA extraction buffer
There is no particular limitation with the consumption of E.C. 3.4.21.64, can be respectively various components commonly used in the art and use
Amount, this is known to those skilled in the art, will not be described here.
In the inventive method, for being processed with DNA extraction buffer, lysozyme and E.C. 3.4.21.64
Method there is no particular limitation, various methods commonly used in the art can be respectively, this be this area skill
Well known to art personnel, will not be described here.
In the inventive method, for the method for DNA purification process, there is no particular limitation, can be this
The method of the conventional various DNA purification process in field, under preferable case, DNA purification process includes：
After E.C. 3.4.21.64 process, centrifuging and taking supernatant, and use chloroform-isoamyl alcohol solution, phenol-chlorine successively
Imitative-isoamyl alcohol, isopropanol and ethanol carries out purification.
In the inventive method, for chloroform-isoamyl alcohol solution, phenol chloroform-isoamyl alcohol, isopropyl
Alcohol and ethanol carry out the method for purification there is no particular limitation, can be respectively various sides commonly used in the art
Method, this are known to those skilled in the art, will not be described here.
In the inventive method, for the method for DNA dissolution process, there is no particular limitation, can be this
The method of the conventional various DNA dissolution process in field, under preferable case, DNA purification process is obtained
DNA precipitations TE for arriving or distilled water dissolving.It will be understood by those skilled in the art that in DNA
After dissolution process, according to actual needs, the DNA of dissolving can be placed in -20 DEG C of preservations first.
Second aspect, the invention provides application of the said method in soil micro-ecosystem analysis.
Embodiment
Hereinafter will be described the present invention by embodiment and comparative example.If no special instructions, make
Each material is commercially available, and the various methods of employing are the conventional method of this area.
Ultraluminescence spectrophotometer is purchased from SHIMADZU companies, model UV2450.
Soil takes from Tsinghua Campus, and collection soil is yellow clay, and sampling depth is 5-10cm tables soil
Layer, is sampled by 5 points of methods, is put in sterile bag after mixing, 4 DEG C of preservations, completes soil microorganism in 24h
The extraction of STb gene.
Soil microorganism total DNA extraction test kit is PowerDNA Isolation Kit, are purchased from
MOBIO companies, article No. are Catalog#12888-50.
Embodiment 1
The method that the present embodiment is used for the extraction soil microorganism STb gene that the present invention is described.
(1) 1g pedotheques are taken and is added in 10mL room temperature centrifuge tubes；
(2) 4mL Polymeric sodium metaphosphate. solution is added in centrifuge tube, stir, then in 10000rpm
Lower room temperature is centrifuged 10min, takes precipitation, and wherein, the concentration of Polymeric sodium metaphosphate. solution is 20g/L, and pH is 8.5；
Repetitive operation 3 times；
(3) 3.5mL Crombach buffer is added, stirring is in the precipitation obtained to step (2)
Even, then room temperature centrifugation 10min under 10000rpm, takes precipitation, wherein, Crombach buffer
Including Tris-HCl and EDTA-Na2, the concentration of Tris-HCl is 0.033mol/L, EDTA-Na2Dense
Spend for 0.001mol/L, the pH of Crombach buffer is 8.0；Repetitive operation 3 times；
(4) 1.5mL isopropanols are added in the precipitation obtained to step (3), is stirred, stood
10min, then room temperature centrifugation 10min under 12000rpm, takes precipitation；Repetitive operation 3 times；
(5) 2mL DNA extraction buffer and 0.5g glass are added in the precipitation obtained to step (4)
Pearl, stirs, and wherein, DNA extraction buffer includes Tris-HCl, EDTA-Na2, sodium phosphate,
The concentration of NaCl and CTAB, Tris-HCl is 0.1mol/L, EDTA-Na2Concentration be 0.1mol/L,
The concentration of sodium phosphate is 0.1mol/L, and the concentration of NaCl is 1.5mol/L, and the concentration of CTAB is 1 mass
%, the pH of DNA extraction buffer is 8.0；
(6) lysozyme soln of 40 μ L 50g/L is added, is vibrated in 37 DEG C, the shaking table of 200rpm
1h；
(7) 20 μ L Proteinase K Solutions of addition, 55 DEG C of water-bath 1h, wherein, Proteinase K Solution bag
E.C. 3.4.21.64, Tris-HCl and calcium acetate is included, the concentration of E.C. 3.4.21.64 is 10g/L, and Tris-HCl's is dense
Spend for 0.05mol/L, the concentration of calcium acetate is 1.5mol/L, and the pH of Proteinase K Solution is 8.0；
(8) under 10000rpm room temperature centrifugation 10min, take supernatant and be added to 5mL room temperatures from
In heart pipe；
(9) 2.5mL chloroform-isoamyl alcohol solution, mix homogeneously are added, agitator vibrates 5min, quiet
Layering is put, wherein, the imitative volume ratio with isoamyl alcohol of chloroform-isoamyl alcohol Chlorine in Solution is 24:1；
(10) careful extract water phase it is added in new 5mL room temperature centrifuge tubes, adds 2.5mL's
Phenol chloroform-isoamyl alcohol, agitator vibration 5min, stratification, wherein, phenol chloroform-different
In amyl alcohol solution, phenol, chloroform, the volume ratio of isoamyl alcohol are 24:24:1；
(11) careful extract water phase it is added in new 15mL room temperature centrifuge tubes, adds 0.6 times of body
Long-pending isopropanol, mixes, and is placed in 4 DEG C of refrigerator 1h, waits white flock precipitate to occur；
(12) room temperature centrifugation 20min under 10000rpm, abandons supernatant, takes precipitation；
(13) ethanol solution that 2mL volume fractions are 70%, resuspended, room under 10000rpm are added
Temperature centrifugation 20min, abandons supernatant, takes precipitation；
(14) repeat upper step, stand, make ethanol volatilize completely；
(15) add 150 μ L TE buffer solution dissolution precipitations and be transferred to clean 1.5mL room temperatures from
In heart pipe, -20 DEG C of preservations, wherein, in TE buffer solution, the concentration of Tris-HCl is 0.01mol/L,
EDTA-Na2Concentration be 0.001M, the pH of TE buffer solution is 8.0.
Microorganism total DNA in three parts of pedotheques is extracted according to step (1)-(15), using ultraviolet glimmering
Light spectrophotometer determines the extracted amount of three parts of samples respectively and characterizes the A of purity260/A280And
A260/A230, averaged.As a result as shown in table 1.
Embodiment 2
The method that the present embodiment is used for the extraction soil microorganism STb gene that the present invention is described.
(1) 1g pedotheques are taken and is added in 10mL room temperature centrifuge tubes；
(2) 2mL Polymeric sodium metaphosphate. solution is added in centrifuge tube, stir, then in 12000rpm
Lower room temperature is centrifuged 8min, takes precipitation, and wherein, the concentration of Polymeric sodium metaphosphate. solution is 25g/L, and pH is 8；
Repetitive operation 4 times；
(3) 6mL Crombach buffer is added in the precipitation obtained to step (2), stir,
Then room temperature centrifugation 10min under 10000rpm, takes precipitation, and wherein, Crombach buffer includes
Tris-HCl and EDTA-Na2, the concentration of Tris-HCl is 0.03mol/L, EDTA-Na2Concentration be
The pH of 0.0005mol/L, Crombach buffer is 7.5；Repetitive operation 2 times；
(4) 3mL isopropanols are added in the precipitation obtained to step (3), are stirred, stand 10min,
Then room temperature centrifugation 10min under 12000rpm, takes precipitation；Repetitive operation 2 times；
(5) 2mL DNA extraction buffer and 0.5g glass are added in the precipitation obtained to step (4)
Pearl, stirs, and wherein, DNA extraction buffer includes Tris-HCl, EDTA-Na2, sodium phosphate,
The concentration of NaCl and CTAB, Tris-HCl is 0.1mol/L, EDTA-Na2Concentration be 0.1mol/L,
The concentration of sodium phosphate is 0.1mol/L, and the concentration of NaCl is 1.5mol/L, and the concentration of CTAB is 1 mass
%, the pH of DNA extraction buffer is 8.0；
(6) lysozyme soln of 40 μ L 50g/L is added, is vibrated in 37 DEG C, the shaking table of 200rpm
1h；
(7) 20 μ L Proteinase K Solutions of addition, 55 DEG C of water-bath 1h, wherein, Proteinase K Solution bag
E.C. 3.4.21.64, Tris-HCl and calcium acetate is included, the concentration of E.C. 3.4.21.64 is 10g/L, and Tris-HCl's is dense
Spend for 0.05mol/L, the concentration of calcium acetate is 1.5mol/L, and the pH of Proteinase K Solution is 8.0；
(8) under 10000rpm room temperature centrifugation 10min, take supernatant and be added to 5mL room temperatures from
In heart pipe；
(9) 2.5mL chloroform-isoamyl alcohol solution, mix homogeneously are added, agitator vibrates 5min, quiet
Layering is put, wherein, the imitative volume ratio with isoamyl alcohol of chloroform-isoamyl alcohol Chlorine in Solution is 24:1；
(10) careful extract water phase it is added in new 5mL room temperature centrifuge tubes, adds 2.5mL's
Phenol chloroform-isoamyl alcohol, agitator vibration 5min, stratification, wherein, phenol chloroform-different
In amyl alcohol solution, phenol, chloroform, the volume ratio of isoamyl alcohol are 24:24:1；
(11) careful extract water phase it is added in new 15mL room temperature centrifuge tubes, adds 0.6 times of body
Long-pending isopropanol, mixes, and is placed in 4 DEG C of refrigerator 1h, waits white flock precipitate to occur；
(12) room temperature centrifugation 20min under 10000rpm, abandons supernatant, takes precipitation；
(13) ethanol solution that 2mL volume fractions are 70%, resuspended, room under 10000rpm are added
Temperature centrifugation 20min, abandons supernatant, takes precipitation；
(14) repeat upper step, stand, make ethanol volatilize completely；
(15) add 150 μ L TE buffer solution dissolution precipitations and be transferred to clean 1.5mL room temperatures from
In heart pipe, -20 DEG C of preservations, wherein, in TE buffer solution, the concentration of Tris-HCl is 0.01mol/L,
EDTA-Na2Concentration be 0.001M, the pH of TE buffer solution is 8.0.
Microorganism total DNA in three parts of pedotheques is extracted according to step (1)-(15), using ultraviolet glimmering
Light spectrophotometer determines the extracted amount of three parts of samples respectively and characterizes the A of purity260/A280And
A260/A230, averaged.As a result as shown in table 1.
Embodiment 3
The method that the present embodiment is used for the extraction soil microorganism STb gene that the present invention is described.
(1) 1g pedotheques are taken and is added in 10mL room temperature centrifuge tubes；
(2) 6mL Polymeric sodium metaphosphate. solution is added in centrifuge tube, stir, then in 10000rpm
Lower room temperature is centrifuged 10min, takes precipitation, and wherein, the concentration of Polymeric sodium metaphosphate. solution is 15g/L, and pH is 9；
Repetitive operation 2 times；
(3) 2mL Crombach buffer is added in the precipitation obtained to step (2), stir,
Then room temperature centrifugation 10min under 10000rpm, takes precipitation, and wherein, Crombach buffer includes
Tris-HCl and EDTA-Na2, the concentration of Tris-HCl is 0.04mol/L, EDTA-Na2Concentration be
The pH of 0.0015mol/L, Crombach buffer is 8.5；Repetitive operation 4 times；
(4) 1mL isopropanols are added in the precipitation obtained to step (3), are stirred, stand 10min,
Then room temperature centrifugation 10min under 12000rpm, takes precipitation；Repetitive operation 4 times；
(5) 2mL DNA extraction buffer and 0.5g glass are added in the precipitation obtained to step (4)
Pearl, stirs, and wherein, DNA extraction buffer includes Tris-HCl, EDTA-Na2, sodium phosphate,
The concentration of NaCl and CTAB, Tris-HCl is 0.1mol/L, EDTA-Na2Concentration be 0.1mol/L,
The concentration of sodium phosphate is 0.1mol/L, and the concentration of NaCl is 1.5mol/L, and the concentration of CTAB is 1 mass
%, the pH of DNA extraction buffer is 8.0；
(6) lysozyme soln of 40 μ L 50g/L is added, is vibrated in 37 DEG C, the shaking table of 200rpm
1h；
(7) 20 μ L Proteinase K Solutions of addition, 55 DEG C of water-bath 1h, wherein, Proteinase K Solution bag
E.C. 3.4.21.64, Tris-HCl and calcium acetate is included, the concentration of E.C. 3.4.21.64 is 10g/L, and Tris-HCl's is dense
Spend for 0.05mol/L, the concentration of calcium acetate is 1.5mol/L, and the pH of Proteinase K Solution is 8.0；
(8) under 10000rpm room temperature centrifugation 10min, take supernatant and be added to 5mL room temperatures from
In heart pipe；
(9) 2.5mL chloroform-isoamyl alcohol solution, mix homogeneously are added, agitator vibrates 5min, quiet
Layering is put, wherein, the imitative volume ratio with isoamyl alcohol of chloroform-isoamyl alcohol Chlorine in Solution is 24:1；
(10) careful extract water phase it is added in new 5mL room temperature centrifuge tubes, adds 2.5mL's
Phenol chloroform-isoamyl alcohol, agitator vibration 5min, stratification, wherein, phenol chloroform-different
In amyl alcohol solution, phenol, chloroform, the volume ratio of isoamyl alcohol are 24:24:1；
(11) careful extract water phase it is added in new 15mL room temperature centrifuge tubes, adds 0.6 times of body
Long-pending isopropanol, mixes, and is placed in 4 DEG C of refrigerator 1h, waits white flock precipitate to occur；
(12) room temperature centrifugation 20min under 10000rpm, abandons supernatant, takes precipitation；
(13) ethanol solution that 2mL volume fractions are 70%, resuspended, room under 10000rpm are added
Temperature centrifugation 20min, abandons supernatant, takes precipitation；
(14) repeat upper step, stand, make ethanol volatilize completely；
(15) add 150 μ L TE buffer solution dissolution precipitations and be transferred to clean 1.5mL room temperatures from
In heart pipe, -20 DEG C of preservations, wherein, in TE buffer solution, the concentration of Tris-HCl is 0.01mol/L,
EDTA-Na2Concentration be 0.001M, the pH of TE buffer solution is 8.0.
Microorganism total DNA in three parts of pedotheques is extracted according to step (1)-(15), using ultraviolet glimmering
Light spectrophotometer determines the extracted amount of three parts of samples respectively and characterizes the A of purity260/A280And
A260/A230, averaged.As a result as shown in table 1.
Embodiment 4
According to the method for embodiment 1, except for the difference that, step (3) is not carried out.
Microorganism total DNA in three parts of pedotheques is extracted according to above-mentioned steps, using Ultraluminescence light splitting
Photometer determines the extracted amount of three parts of samples respectively and characterizes the A of purity260/A280And A260/A230, ask for
Meansigma methodss.As a result as shown in table 1.
Embodiment 5
According to the method for embodiment 1, except for the difference that, 1 operation in step (2), is only carried out.
Microorganism total DNA in three parts of pedotheques is extracted according to above-mentioned steps, using Ultraluminescence light splitting
Photometer determines the extracted amount of three parts of samples respectively and characterizes the A of purity260/A280And A260/A230, ask for
Meansigma methodss.As a result as shown in table 1.
Embodiment 6
According to the method for embodiment 1, except for the difference that, 1 operation in step (3), is only carried out.
Microorganism total DNA in three parts of pedotheques is extracted according to above-mentioned steps, using Ultraluminescence light splitting
Photometer determines the extracted amount of three parts of samples respectively and characterizes the A of purity260/A280And A260/A230, ask for
Meansigma methodss.As a result as shown in table 1.
Embodiment 7
According to the method for embodiment 1, except for the difference that, 1 operation in step (4), is only carried out.
Microorganism total DNA in three parts of pedotheques is extracted according to above-mentioned steps, using Ultraluminescence light splitting
Photometer determines the extracted amount of three parts of samples respectively and characterizes the A of purity260/A280And A260/A230, ask for
Meansigma methodss.As a result as shown in table 1.
Embodiment 8
According to the method for embodiment 1, except for the difference that, Crombach buffer solution soil is first used, then
Soil is washed with isopropanol, finally soil is washed with Polymeric sodium metaphosphate. solution, i.e., first carry out in embodiment 1
Step (3), then step (4) is carried out, finally carry out step (2).
Microorganism total DNA in three parts of pedotheques is extracted according to above-mentioned steps, using Ultraluminescence light splitting
Photometer determines the extracted amount of three parts of samples respectively and characterizes the A of purity260/A280And A260/A230, ask for
Meansigma methodss.As a result as shown in table 1.
Comparative example 1
The total of soil microorganism in pedotheque same as Example 1 is extracted using SDS-CTAB cracking processs
DNA, concrete grammar include：5g pedotheques are weighed in mortar, is extracted with 20.0mL phosphate
Buffer (0.25mol/L NaH2PO4, 0.1mol/L Tris-HCl (pH 8.0), 0.1mol/L EDTA
(pH 8.0), 1%CTAB (w/v)；Autoclaving, using 1% beta -mercaptoethanol of front addition (v/v)) grind
It is placed in after mill in 50mL sterile centrifugation tubes, adds 4.0mL 20%SDS (w/v), vortex 2min, so
68 DEG C of water-bath 2h, and every 15min afterwards turn upside down and mix for several times.Afterwards, 13000rpm centrifugations
15min；Supernatant is transferred to another new pipe, 500 μ L 5mol/L KAc solution and 1.67mL is added
PEG8000, places 15min at -20 DEG C, and 13000rpm is centrifuged 15min, abandons supernatant, with 500 μ L
The resuspended precipitations of 2 × CTAB, 68 DEG C of water-bath 1h add equal-volume chloroform/isoamyl alcohol (chloroform and isoamyl alcohol
Volume ratio is 24：1), vibration is mixed, and stands 5min, is centrifuged 15min under 4 DEG C, 13000rpm；
Supernatant is transferred to another new pipe, equal-volume isopropanol is added, is mixed, under room temperature, place 15min,
13000rpm is centrifuged 15min, is washed 2 times with 70% ethanol (v/v), air-dries, is dissolved in 50 μ L TE
Buffer solution (0.01mol/L Tris-HCl, 0.001M EDTA-Na2, pH be 8.0) in, at 4 DEG C protect
Deposit standby.
Microorganism total DNA in three parts of pedotheques is extracted using SDS-CTAB cracking processs, using ultraviolet
Spectrofluorophotometer determines the extracted amount of three parts of samples respectively and characterizes the A of purity260/A280And
A260/A230, averaged.As a result as shown in table 1.
Comparative example 2
Extracted according to the operating procedure of its description using soil microorganism total DNA extraction test kit and real
Apply the STb gene of soil microorganism in 1 identical pedotheque of example.
Microbial total in three parts of pedotheques is extracted using soil microorganism total DNA extraction test kit
DNA, determines the extracted amount of three parts of samples respectively using Ultraluminescence spectrophotometer and characterizes purity
A260/A280And A260/A230, averaged.As a result as shown in table 1.
Comparative example 3
According to the method for embodiment 1, except for the difference that, with the inclined phosphorus containing PVP-K30 in step (2)
(i.e. the Polymeric sodium metaphosphate. solution contains Polymeric sodium metaphosphate. and PVP-K30 to acid sodium solution, and the concentration of Polymeric sodium metaphosphate. is
The concentration of 20g/L, PVP-K30 is 10g/L, and the pH of the Polymeric sodium metaphosphate. solution replaces reality for 8.5)
The Polymeric sodium metaphosphate. solution in example 1 is applied, and does not carry out step (3) and step (4).
Microorganism total DNA in three parts of pedotheques is extracted according to above-mentioned steps, using Ultraluminescence light splitting
Photometer determines the extracted amount of three parts of samples respectively and characterizes the A of purity260/A280And A260/A230, ask for
Meansigma methodss.As a result as shown in table 1.
Comparative example 4
According to the method for embodiment 1, except for the difference that, step (4) is not carried out.
Microorganism total DNA in three parts of pedotheques is extracted according to above-mentioned steps, using Ultraluminescence light splitting
Photometer determines the extracted amount of three parts of samples respectively and characterizes the A of purity260/A280And A260/A230, ask for
Meansigma methodss.As a result as shown in table 1.
Table 1
| Extracted amount (μ g/g) | A260/A280 | A260/A230 | |
| Embodiment 1 | 78.6 | 1.68 | 1.75 |
| Embodiment 2 | 77.4 | 1.65 | 1.70 |
| Embodiment 3 | 77.9 | 1.66 | 1.72 |
| Embodiment 4 | 69.4 | 1.45 | 1.56 |
| Embodiment 5 | 76.2 | 1.51 | 1.51 |
| Embodiment 6 | 76.6 | 1.56 | 1.68 |
| Embodiment 7 | 76.9 | 1.60 | 1.65 |
| Embodiment 8 | 77.0 | 1.64 | 1.70 |
| Comparative example 1 | 6.4 | 1.62 | 1.46 |
| Comparative example 2 | 4.4 | 1.69 | 0.44 |
| Comparative example 3 | 62.4 | 1.41 | 1.40 |
| Comparative example 4 | 60.2 | 1.40 | 1.33 |
Embodiment in table 1 is compared with the data of comparative example and is understood, the extraction soil microorganism of the present invention is total
The method of DNA, can effectively extract the STb gene of soil microorganism, and can significantly improve and obtain
The yield and purity of DNA.
Embodiment 1 in table 1 is compared with the data of embodiment 4 and is understood, when pretreatment is carried out to soil,
Except washing soil with Polymeric sodium metaphosphate. solution and being washed in addition to soil with isopropanol, while slow with Crombach
Liquid washing soil is rushed, the yield and purity of the DNA for obtaining can be further improved.
Embodiment 1 in table 1 is compared with the data of embodiment 5 and is understood, soil is washed with Polymeric sodium metaphosphate. solution
When the number of times of earth is 2-4, the yield and purity of the DNA for obtaining can be further improved.
Embodiment 1 in table 1 is compared with the data of embodiment 6 and is understood, washed with Crombach buffer
When the number of times for washing soil is 2-4, the yield and purity of the DNA for obtaining can be further improved.
Embodiment 1 in table 1 being compared with the data of embodiment 7 and being understood, the secondary of soil is washed with isopropanol
When number is 2-4, the yield and purity of the DNA for obtaining can be further improved.
Embodiment 1 in table 1 is compared with the data of embodiment 8 and is understood, according to molten with Polymeric sodium metaphosphate. successively
When the mode of liquid, Crombach buffer and isopropanol washing soil carries out pretreatment to soil, can
The yield and purity of the DNA that obtain further are improved.
The preferred embodiment of the present invention described in detail above, but, the present invention is not limited to above-mentioned reality
The detail in mode is applied, in the range of the technology design of the present invention, can be to the technical side of the present invention
Case carries out multiple simple variants, and these simple variants belong to protection scope of the present invention.
It is further to note that each particular technique described in above-mentioned specific embodiment is special
Levy, in the case of reconcilable, can be combined by any suitable means, in order to avoid need not
The repetition that wants, the present invention are no longer separately illustrated to various possible compound modes.
Additionally, combination in any can also be carried out between a variety of embodiments of the present invention, as long as its
Without prejudice to the thought of the present invention, which should equally be considered as content disclosed in this invention.
Claims (11)
1. a kind of method for extracting soil microorganism STb gene, methods described includes soil microorganism cell
Break process, DNA purification process and DNA dissolution process, it is characterised in that thin in soil microorganism
Pretreatment is carried out before born of the same parents' break process to soil, and the pretreatment includes washing soil with Polymeric sodium metaphosphate. solution
Earth and soil is washed with isopropanol.
2. method according to claim 1, wherein, the pretreatment also includes using Crombach
Buffer solution soil, it is preferable that the pretreatment includes using Polymeric sodium metaphosphate. solution, Crombach successively
Buffer and isopropanol washing soil.
3. method according to claim 1, wherein, washs the secondary of soil with Polymeric sodium metaphosphate. solution
Number is 2-4.
4. method according to claim 1, wherein, the number of times for washing soil with isopropanol is 2-4.
5. method according to claim 2, wherein, with Crombach buffer solution soil
Number of times is 2-4.
6. method according to claim 1, wherein, the concentration of the Polymeric sodium metaphosphate. solution is
15-25g/L, pH are 8-9；Preferably, relative to every gram of soil, the consumption of the Polymeric sodium metaphosphate. solution
For 2-6mL.
7. method according to claim 1, wherein, relative to every gram of soil, the use of isopropanol
Measure as 1-3mL.
8. method according to claim 1, wherein, the Crombach buffer includes
Tris-HCl and EDTA-Na2, the concentration of the Tris-HCl is 0.03-0.04mol/L, described
EDTA-Na2Concentration be 0.0005-0.0015mol/L, the pH of the Crombach buffer is
7.5-8.5；Preferably, relative to every gram of soil, the consumption of the Crombach buffer is 2-6mL.
9. method according to claim 1 and 2, wherein, the soil microorganism cell breakage
Process includes：DNA extraction buffer, lysozyme and egg is sequentially added in the sample that pretreatment is obtained
White enzyme K process.
10. method according to claim 9, wherein, the DNA purification process includes：?
After E.C. 3.4.21.64 process, centrifuging and taking supernatant, and use chloroform-isoamyl alcohol solution, phenol chloroform successively
- isoamyl alcohol, isopropanol and ethanol carry out purification.
Application of the method in 11. claim 1-10 described in any one in soil micro-ecosystem analysis.
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