CN104178481A - Kit for extracting soil microbial genome DNA within 30 minute - Google Patents
Kit for extracting soil microbial genome DNA within 30 minute Download PDFInfo
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- CN104178481A CN104178481A CN201410449878.3A CN201410449878A CN104178481A CN 104178481 A CN104178481 A CN 104178481A CN 201410449878 A CN201410449878 A CN 201410449878A CN 104178481 A CN104178481 A CN 104178481A
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Abstract
The invention provides a kit for extracting soil microbial genome DNA within 30 minutes. The kit provided by the invention is capable of fast and efficiently extracting the soil microbial genome DNA with high purity and high concentration within 30 minutes, and thus overcoming the defects that the traditional soil microbial genome DNA extraction is long in time consumption and needs further purification. The soil microbial genome DNA extracted by utilizing the kit can be directly used for a downstream experiment without being further purified. The kit mainly comprises pretreatment of a soil sample and extraction and purification of the soil microbial genome DNA. The method is short in time consumption and high in efficiency.
Description
Technical field
The invention provides the test kit that extracts soil microbe genome DNA in a kind of 30 minutes, belong to biological technical field.
Technical background
Soil is except providing nutrition source and moisture for terrestrial plant, or plant-growth, the main place of carrying out photosynthesis, energy exchange; Also have microorganism growth needed all nutritive substances of breeding and various condition, be the good life area of microorganism simultaneously, has the title of " natural medium of microorganism ".
Soil is the main habitat of microorganism, is heterogeneous system the most complicated in physical environment, and this has determined the variation of Soil Micro-environment and the height diversity of soil microorganisms.Every gram of soil contains about 10
7individual prokaryote, but only have 0.1%～10% soil microorganisms to cultivate.In microbiological research field because more than 99% microorganism be all at present not (difficulty) by pure culture, past people concentrate on less than on 1% microorganism substantially to the understanding of microbial world 1.To these not the research of culturing micro-organisms diversity and community function will greatly expand our understanding to life, comprise how understand life tolerates the interaction between extreme environment, new bioenergy, life evolution and microorganism and environment.
Microbial environment genomics has been brought unprecedented opportunities for excavating to greatest extent Microbial resources, has become international Life Sci-Tech and has researched and developed most important focus and forward position.For fully realize can not culturing micro-organisms affluent resources, in conjunction with the development of modern molecular biology technique, set up many needs microorganism carried out to independent novel method and new technologies of cultivating.If denaturing gradient gel electrophoresis (DGGE) method is that target gene is carried out to pcr amplification, by PCR product being carried out to the composition information of separated and identification and analysis structure of soil microbial community; Random amplified polymorphic DNA technique (RAPD) is that the different random primer of application carries out pcr amplification under non-strict condition, by analyzing the band polymorphism of PCR product in gel electrophoresis, reflects the diversity of microorganism in sample.
About the existing many research reports of the structure of soil microorganisms genomics technology, main breakthrough is to need enough high-quality DNA, therefore directly from edatope, extracts and purify DNA is the step of most critical wherein.The research of soil microorganisms genomics technology aspect China's soil and environmental microorganism ecology is ground zero, to the tracking of this new technology and application, will effectively promote China's Ecological Distribution of Soil Microorganisms to learn the development of research.Therefore, study a kind of test kit of high efficiency extraction soil microbe genome DNA, for carrying out on a large scale the research of soil microorganisms genomics, seem very necessary.
summary of the invention:
The invention provides the test kit that extracts soil microbe genome DNA in a kind of 30 minutes, utilize this test kit can be efficiently, rapid extraction soil microbial DNA, extract that the DNA purity obtaining is high, concentration is high.
A test kit for rapid extraction soil microbe genome DNA in 30 minutes, described test kit comprises:
(1) configuration of Buffer A solution: pH=5-6,0.5-2M Tris-HCl;
(2) configuration of Buffer B solution: 0.1-0.3M Al
2(SO
4)
3;
(3) configuration of Buffer C solution: 3-5M NaOH;
(4) configuration of Buffer D solution: pH=7-9,0.1-0.2M Tris-HCl;
(5) configuration of Buffer E solution: 300-350 μ l, 10wt.%SDS;
(6) configuration of Buffer F solution: take 2-3g LiCl, 1-2g Tris, 3-4g EDTA, adds distilled water to dissolve and is adjusted to pH=7.5-8.5, is settled to 100ml;
(7) protein liquid removal solution preparation: 4-6M Guanidinium hydrochloride, 15-25 mM Tris-HCl, pH6.5-7.0, adds ethanol before use, and alcohol concn is 35-40wt.%;
(8) rinsing liquid solution preparation: 15-25mM NaCl, 1-3mM Tris-HCl, pH7.0-8.0;
(9) eluate solution preparation: 5-15mM Tris-HCl, pH8.0-9.0.
The screening formulation of mentioned reagent box:
(1) configuration of Buffer A solution: pH=5.5,1M Tris-HCl;
(2) configuration of Buffer B solution: 0.2M Al
2(SO
4)
3;
(3) configuration of Buffer C solution: 4M NaOH;
(4) configuration of Buffer D solution: pH=8,0.1M Tris-Tris-HCl;
(5) configuration of Buffer E solution: 325 μ l, 10%SDS;
(6) configuration of Buffer F solution: take 2.416g LiCl, 1.211g Tris, 3.506g EDTA, adds distilled water to dissolve and is adjusted to pH=8.0, is settled to 100ml;
(7) protein liquid removal solution preparation: 5M Guanidinium hydrochloride, 20 mM Tris-HCl, pH6.6, adds ethanol before use, and alcohol concn is 38wt.%;
(8) rinsing liquid solution preparation: 20mM NaCl, 2mM Tris-HCl, pH7.5;
(9) eluate solution preparation: 10mM Tris-HCl, pH 8.5.
Described test kit extracts the method for soil microbe genome DNA, comprises the pre-treatment of pedotheque, the extraction of soil microbe genome DNA and purifying.Concrete steps are:
(1)
the pre-treatment of pedotheque:weigh in the balance get 0.5g soil in the centrifuge tube of 2ml, add 100 μ l Buffer A, 600 μ l sterilized waters, 300 μ l Buffer B, vortex concussion 30s; Add 100 μ l Buffer C, 300 μ lBuffer D, vortex concussion 30s; The centrifugal 2min of 8000g, removes supernatant;
(2) extraction of soil microbe genome DNA and purifying:add 325 μ l Buffer D, 0.5g 0.5mm granulated glass sphere, 325 μ l Buffer E, 325 μ l Buffer F, vortex concussion 1min, 4 ℃ of centrifugal 2min of 11000g, draw 750ul supernatant to 1.5ml centrifuge tube, add 750 μ l phenol: chloroform: primary isoamyl alcohol=25:24:1, vortex concussion 30s, 4 ℃ of centrifugal 1min of 16000g, supernatant is transferred to 1.5ml centrifuge tube, add isopyknic precipitation agent, sample is added on silica-based plasma membrane nucleic acid purification post, the centrifugal 30s of 12000 g, abandon waste liquid, add 500 μ l protein liquid removals, the centrifugal 30s of 12000 g, add 500 μ l rinsing liquids, the centrifugal 30s of 12000 g, dry up, add 50 μ l elutriants, electrophoresis detection in 1% sepharose.
The purity 1.78 of the soil microorganisms of gained.The soil microbial DNA extracting is carried out to the experiment of 16sr DNA cloning, find that the soil microbial DNA that present method obtains does not need can amplify 16sr DNA through being further purified just, this has further proved that present method can obtain the soil microbial DNA that purity is high.
Remarkable advantage of the present invention:
1. be applicable to various different soils samples.
2. only need within 30 minutes, just can from soil, extract microbe genome DNA.
3. the soil microbe genome DNA extracting has higher purity, is applicable to next step experiment.
Accompanying drawing explanation
Fig. 1 is that the microbe genome DNA of 5 kinds of different soils samples extracts result figure; Swimming lane 1,2,3,4,5 represent respectively sugarcane soil microbe genome DNA, peanut soil microbe genome DNA, corn soil microbe genome DNA, allelopathic paddy rice PI312777 soil microbe genome DNA, non-allelopathic oryza sativa l. emont soil microbe genome DNA.
Fig. 2 is the microbial genome 16s rDNA amplification figure of 5 kinds of different soils samples; Swimming lane 1,2,3,4,5 represent respectively sugarcane soil microorganisms genome 16s rDNA amplification figure, peanut soil microorganisms genome 16s rDNA amplification figure, corn soil microorganisms genome 16s rDNA amplification figure, allelopathic paddy rice PI312777 soil microorganisms genome 16s rDNA amplification figure, non-allelopathic oryza sativa l. emont soil microorganisms genome 16s rDNA amplification figure.
Specific embodiment
embodiment 1
Test kit and the method for rapid extraction soil microbe genome DNA in a kind of 30 minutes of the present invention, concrete steps are as follows:
(1) raw material: fresh cane rhizosphere soil, the raw rhizosphere soil of fresh flowers, fresh corn rhizosphere soil, fresh allelopathic paddy rice PI312777 rhizosphere soil, fresh non-allelopathic oryza sativa l. emont rhizosphere soil.
(2) pre-treatment of pedotheque: take respectively different pedotheque 0.5g in the centrifuge tube of 2ml with balance, add 100 μ l Buffer A, 600 μ l sterilized waters, 300 μ l Buffer B, vortex concussion 30s; Add 100 μ l Buffer C, 300 μ lBuffer D, vortex concussion 30s; The centrifugal 2min of 8000g, removes supernatant.
(3) extraction of soil microbe genome DNA and purifying: add 325 μ l Buffer D, 0.5g 0.5mm granulated glass sphere, 325 μ l Buffer E, 325 μ l Buffer F, vortex concussion 1min,, 4 ℃ of centrifugal 2min of 11000g.Draw 750ul supernatant to 1.5ml centrifuge tube, add 750 μ l phenol: chloroform: primary isoamyl alcohol (25:24:1), vortex concussion 30s, 4 ℃ of centrifugal 1min of 16000g.Supernatant is transferred to 1.5ml centrifuge tube, add isopyknic precipitation agent, sample is added on silica-based plasma membrane nucleic acid purification post, and the centrifugal 30s of 12000 g, abandons waste liquid, add 500 μ l protein liquid removals, the centrifugal 30s of 12000 g, adds 500 μ l rinsing liquids, the centrifugal 30s of 12000 g, dry up, add 50 μ l elutriants.Electrophoresis detection in 1% sepharose.
(4) amplification soil microorganisms 16sr DNA gene: carry out the amplification of soil microorganisms 16sr DNA gene by the mode of PCR, pcr amplification system is as follows: rTaq enzyme 0.3 μ l, PCR buffer 2.5 μ l, DNTP 1 μ l, primer R 1 μ l, primers F 1 μ l, soil microbe genome DNA 1 μ l, aqua sterilisa 18.2 μ l.Pcr amplification program is as follows: 95 ° of C 5min; 95 ° of C 30s, 58 ° of C 30s, 72 ° of C 30s, 35 circulations; 72 ° of C 5min.Electrophoresis detection in 1% sepharose for the product that amplification is obtained.
Test kit formula is:
(1) configuration of Buffer A solution: 1M Tris-Cl (pH=5.5);
(2) configuration of Buffer B solution: 0.2M Al
2(SO
4)
3;
(3) configuration of Buffer C solution: 4M NaOH;
(4) configuration of Buffer D solution: 0.1M Tris-Cl (pH=8);
(5) configuration of Buffer E solution: 325 μ l 10%SDS;
(6) configuration of Buffer F solution: take 2.416g LiCl, 1.211g Tris, 3.506g EDTA, adds the two of proper volume steam (7) water dissolution and regulate pH=8.0, is settled to 100ml.
(8) protein liquid removal solution preparation: 5M Guanidinium hydrochloride, 20 mM Tris-HCI, pH6.6, adds ethanol before use, and alcohol concn is 38%.
(9) rinsing liquid solution preparation: 20mM NaCL, 2mM Tris-HCI, pH7.5.
(10) eluate solution preparation: 10mM Tris-HCI, pH 8.5.
(11) primer R:5 '-GAGAGTTTGATCCTGGCTCAG-3 '; Primers F: 5 '-GGTTACCTTGTTACGACTT-3 '.
Soil microbe genome DNA detected result is shown in Fig. 1, swimming lane 1,2,3,4,5 represent respectively sugarcane soil microbe genome DNA, peanut soil microbe genome DNA, corn soil microbe genome DNA, allelopathic paddy rice PI312777 soil microbe genome DNA, non-allelopathic oryza sativa l. emont soil microbe genome DNA.The microbe genome DNA band that extraction obtains is clear obviously, without assorted band.Obtained soil microbial DNA is carried out to the amplification of 16sr DNA gene, the results are shown in Figure 2, swimming lane 1,2,3,4,5 represent respectively sugarcane soil microorganisms genome 16s rDNA amplification figure, peanut soil microorganisms genome 16s rDNA amplification figure, corn soil microorganisms genome 16s rDNA amplification figure, allelopathic paddy rice PI312777 soil microorganisms genome 16s rDNA amplification figure, non-allelopathic oryza sativa l. emont soil microorganisms genome 16s rDNA amplification figure.As can be seen from Figure, the DNA proposing by this test kit is without directly carrying out 16s rDNA amplification through being further purified just.
The foregoing is only preferred embodiment of the present invention, all equalizations of doing according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.
SEQUENCE LISTING
<110> Fujian Normal University
Mono-kind of <120> extracts the test kit of soil microbe genome DNA in 30 minutes
<130> 2
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213> artificial sequence
<400> 1
gagagtttga tcctggctca g 21
<210> 2
<211> 19
<212> DNA
<213> artificial sequence
<400> 2
ggttaccttg ttacgactt 19
Claims (4)
1. a test kit for rapid extraction soil microbe genome DNA in 30 minutes, is characterized in that: described test kit comprises:
(1) configuration of Buffer A solution: pH=5-6,0.5-2M Tris-HCl;
(2) configuration of Buffer B solution: 0.1-0.3M Al
2(SO
4)
3;
(3) configuration of Buffer C solution: 3-5M NaOH;
(4) configuration of Buffer D solution: pH=7-9,0.1-0.2M Tris-HCl;
(5) configuration of Buffer E solution: 300-350 μ l, 10wt.%SDS;
(6) configuration of Buffer F solution: take 2-3g LiCl, 1-2g Tris, 3-4g EDTA, adds distilled water to dissolve and is adjusted to pH=7.5-8.5, is settled to 100ml;
(7) protein liquid removal solution preparation: 4-6M Guanidinium hydrochloride, 15-25 mM Tris-HCl, pH6.5-7.0, adds ethanol before use, and alcohol concn is 35-40wt.%;
(8) rinsing liquid solution preparation: 15-25mM NaCl, 1-3mM Tris-HCl, pH7.0-8.0;
(9) eluate solution preparation: 5-15mM Tris-HCl, pH8.0-9.0.
2. the test kit of rapid extraction soil microbe genome DNA in a kind of 30 minutes according to claim 1, is characterized in that: described test kit comprises as follows:
(1) configuration of Buffer A solution: pH=5.5,1M Tris-HCl;
(2) configuration of Buffer B solution: 0.2M Al
2(SO
4)
3;
(3) configuration of Buffer C solution: 4M NaOH;
(4) configuration of Buffer D solution: pH=8,0.1M Tris-Tris-HCl;
(5) configuration of Buffer E solution: 325 μ l, 10%SDS;
(6) configuration of Buffer F solution: take 2.416g LiCl, 1.211g Tris, 3.506g EDTA, adds distilled water to dissolve and is adjusted to pH=8.0, is settled to 100ml;
(7) protein liquid removal solution preparation: 5M Guanidinium hydrochloride, 20 mM Tris-HCl, pH6.6, adds ethanol before use, and alcohol concn is 38wt.%;
(8) rinsing liquid solution preparation: 20mM NaCl, 2mM Tris-HCl, pH7.5;
(9) eluate solution preparation: 10mM Tris-HCl, pH 8.5.
3. test kit as claimed in claim 1 or 2 extracts a method for soil microbe genome DNA, it is characterized in that: described method comprises the pre-treatment of pedotheque the extraction of soil microbe genome DNA and purifying.
4. test kit according to claim 3 extracts the method for soil microbe genome DNA, it is characterized in that: concrete steps are:
(1) pre-treatment of pedotheque: weigh in the balance get 0.5g soil in the centrifuge tube of 2ml, add 100 μ l Buffer A, 600 μ l sterilized waters, 300 μ l Buffer B, vortex concussion 30s; Add 100 μ l Buffer C, 300 μ l Buffer D, vortex concussion 30s; The centrifugal 2min of 8000g, removes supernatant;
(2) extraction of soil microbe genome DNA and purifying: add 325 μ l Buffer D, 0.5g 0.5mm granulated glass sphere, 325 μ l Buffer E, 325 μ l Buffer F, vortex concussion 1min, 4 ℃ of centrifugal 2min of 11000g, draw 750ul supernatant to 1.5ml centrifuge tube, add 750 μ l phenol: chloroform: primary isoamyl alcohol=25:24:1, vortex concussion 30s, 4 ℃ of centrifugal 1min of 16000g, supernatant is transferred to 1.5ml centrifuge tube, add isopyknic precipitation agent, sample is added on silica-based plasma membrane nucleic acid purification post, the centrifugal 30s of 12000 g, abandon waste liquid, add 500 μ l protein liquid removals, the centrifugal 30s of 12000 g, add 500 μ l rinsing liquids, the centrifugal 30s of 12000 g, dry up, add 50 μ l elutriants, electrophoresis detection in 1% sepharose.
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Cited By (5)
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| CN104531680A (en) * | 2014-12-29 | 2015-04-22 | 福建师范大学 | Kit and extraction method for quickly extracting microbial genome DNA from animal fecal microorganisms |
| CN104560955A (en) * | 2014-12-29 | 2015-04-29 | 福建师范大学 | Method for extracting genomic DNA of soil microorganism by using CTAB(cetyl trimethyl ammonium bromide) |
| CN104560953A (en) * | 2014-12-29 | 2015-04-29 | 福建师范大学 | Kit for rapidly extracting sludge microbial genome DNA and extracting method |
| CN104975004A (en) * | 2015-07-28 | 2015-10-14 | 福建师范大学 | Kit for rapidly extracting genomes of animal excrement by virtue of CTAB method |
| CN106399297A (en) * | 2016-09-07 | 2017-02-15 | 贵州茅台酒股份有限公司 | Method for economically and rapidly extracting microbial genome DNA in fermented grains |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104531680A (en) * | 2014-12-29 | 2015-04-22 | 福建师范大学 | Kit and extraction method for quickly extracting microbial genome DNA from animal fecal microorganisms |
| CN104560955A (en) * | 2014-12-29 | 2015-04-29 | 福建师范大学 | Method for extracting genomic DNA of soil microorganism by using CTAB(cetyl trimethyl ammonium bromide) |
| CN104560953A (en) * | 2014-12-29 | 2015-04-29 | 福建师范大学 | Kit for rapidly extracting sludge microbial genome DNA and extracting method |
| CN104560955B (en) * | 2014-12-29 | 2017-08-25 | 福建师范大学 | A kind of method that utilization CTAB extracts soil microbe genome DNA |
| CN104975004A (en) * | 2015-07-28 | 2015-10-14 | 福建师范大学 | Kit for rapidly extracting genomes of animal excrement by virtue of CTAB method |
| CN106399297A (en) * | 2016-09-07 | 2017-02-15 | 贵州茅台酒股份有限公司 | Method for economically and rapidly extracting microbial genome DNA in fermented grains |
| CN106399297B (en) * | 2016-09-07 | 2020-02-14 | 贵州茅台酒股份有限公司 | Method for economically and rapidly extracting microbial genome DNA (deoxyribonucleic acid) in fermented grains |
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Application publication date: 20141203 |