CN1990863A - Small quality fast extraction method for soil total DNA - Google Patents
Small quality fast extraction method for soil total DNA Download PDFInfo
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- CN1990863A CN1990863A CN 200510120584 CN200510120584A CN1990863A CN 1990863 A CN1990863 A CN 1990863A CN 200510120584 CN200510120584 CN 200510120584 CN 200510120584 A CN200510120584 A CN 200510120584A CN 1990863 A CN1990863 A CN 1990863A
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Abstract
The invention discloses a method for fast extracting the total soil DNA. It takes soil as raw material, shaking with quartz sand and silicon dioxide powder, cracking with cetyltrimethylammonium bromide for DNA releasment, the released DNA can be absorbed by diatomite under weak acid or netural pH condition and high concentration thiocyanate ion, and the DNA will be eluted by low salty buffer solution or deionized water under weak alkaline condition. The extracted DNA with enough purity and amount can be used for PCR and endonuclease reaction. The main steps comprise cell cracking, DNA extraction and DNA purification. The invention is characterized by the simple, fast and easy method, and high purity and low cost extracted DNA. The extracted DNA can be used for kit production. The soil total DNA can be extracted in half hour for microbiological and molecular biological research.
Description
Technical field
The invention belongs to microbiology and technical field of molecular biology, be specifically related to the method for the total DNA of a small amount of rapid extraction from soil.
Background technology
The method that is generally used for the soil total DNA extraction at present mainly contains indirect method (Andrew E.Berry, Claudia Chiocchini, TinaSelby, Margherita Sosio, Elizabeth M.H.Wellington. (2003) Isolation of high molecular weight DNA from soil forcloning into BAC vectors.FEMS Microbiology Letters.223:15-20), direct method (Zhou, J., Mary Ann Bruns, M.A., andTiedje, M.J. (1996) DNA Recovery from Soils of Diverse Composition.Applied and Environmental Microbiology62 (2): 316-322) and test kit method (the FastDNA SPIN Kit for Soil of Q.BIOgene company), Chinese invention patent ublic specification of application (publication number CN1456684A, number of patent application: 03120964.5) disclose the design of primers scheme of microflora " a kind of PCR-DGGE research environment ", its method of extracting the total DNA of soil also is a direct method.The indirect method of extracting total DNA from soil is the cracking again of first isolated cell, because many microorganisms and soil particle are combined closely, cell is not easily separated, causes final DNA to yield poorly, and also very time-consuming, this method is generally used less; Direct method is direct lysing cell from soil, and this method is time-consuming and effect is general; The test kit method has simply, fast, but cost an arm and a leg, uneconomical to the extraction of gross sample.
Summary of the invention
The objective of the invention is to overcome the defective that existing method exists, set up a kind of novel method of simple to operate, quick, convenient and price economy.At present domestic also do not have soil DNA to extract test kit, and this method can be used for making test kit, extracts the purer total DNA of soil about half an hour, and directly the substrate of cutting as template and the enzyme of PCR is used for soil microbiology and molecular biological operation.
The present invention is achieved through the following technical solutions:
The applicant has invented the total DNA of a kind of soil novel method of rapid extraction in a small amount.With soil is material, take the common lysing cell released dna of physics and chemical process (being quartz sand, SiO 2 powder vibration and cetyl trimethylammonium bromide (CTAB) coupling), the DNA of release is at slightly acidic or partial neutral pH value and high density thiocyanate ion (SCN
-) down can be by diatomite (main component is a silicon-dioxide) absorption, behind condition changing, DNA is come out by low salt buffer or deionized water wash-out again under weakly alkaline, and the DNA that obtains like this has enough purity and amount is used for PCR and endonuclease reaction.
The present invention includes following steps:
Key step of the present invention comprises: lysis, DNA extracting and purifying.Its concrete steps are as follows:
(1) makes cracking tube: take by weighing each 10 gram of quartz sand and SiO 2 powder, earlier with rare nitric acid washing, then with deionized water wash and soaked overnight, again 80 ℃ of oven dry down, accurately take by weighing quartz sand and each 0.3 gram of SiO 2 powder of oven dry, pack into the centrifuge tube of 2ml, 121 ℃ of sterilizations 30 minutes;
(2) take by weighing soil sample 0.5 and restrain in the cracking tube of step (1), add the lysis buffer (preparation of lysis buffer: 0.1M Tris-HCl, 0.1M EDTA, 0.1MH
3PO
4Buffer, 1.5M NaCl, pH8.0,121 ℃ of sterilizations added CTAB after 30 minutes, making its final concentration is 1% (grams per milliliter)) mixing after 1 milliliter;
(3) with adhesive tape cracking tube is fixed on vortex mixed instrument (originate from its woods Bel instrument Manufacturing Co., Ltd of Haimen City, Jiangsu Province, product type is the QL-901 type) and goes up vibration 5 minutes;
(4) after under 10,000 * g centrifugal 1 minute supernatant liquor as much as possible is being gone in 1.5 milliliters of centrifuge tubes of a cleaning;
(5) 10, under 000 * g after centrifugal 2 minutes careful 0.4 milliliter of the supernatant liquor drawn in 2 milliliters of centrifuge tubes of a cleaning, with (the preparation of adsorbing base suspension: 0.3M HAc Buffer of adsorbing base suspension, 7M KSCN, about pH6, add with the washing of rare nitric acid and deionized water soaked overnight and at 80 ℃ of diatomite of drying down, making its final concentration is 5% (grams per milliliter) again) add 1 milliliter after shaking up;
(6) manually shake up 2 minutes gently, DNA is fully combined with adsorbing base;
(7) 0.8 milliliter of careful abandoning supernatant after under 10,000 * g centrifugal 10 seconds is all drawn the adsorbing base back that suspends again, moves on on the centrifugal purification post (the centrifugal purification post is bought from match Parkson, Shanghai gene engineering company limited) that contains collection tube;
(8) centrifugal 1 minute collecting precipitation under 10,000 * g removes the waste liquid in the collection tube;
(9) on the centrifugal purification post, add 0.5 milliliter of washings (preparation of washings: 12ml 3M NaAc mixes back adjust pH to 7.0 with 100ml ethanol), 10, centrifugal 1 minute washing matrix under 000 * g, remove the waste liquid in the collection tube, again under 10,000 * g centrifugal 2 minutes to remove residual washings;
(10) the centrifugal purification post is gone in 1.5 milliliters of centrifuge tubes of a cleaning, drying is 2 minutes under room temperature;
(11) in the middle of the centrifugal purification post, add the 100 microlitre TE damping fluid (preparations of TE damping fluid: 10mM Tris-HCl, 1mM EDTA, pH8.0, sterilized 30 minutes for 121 ℃) or deionized water, with 1 minute abundant wash-out of light finger bomb tube wall with assurance DNA, centrifugal 1 minute collection DNA under 10,000 * g;
(12) DNA that extracts with step (11) is a template, with 16S rDNA primer 338F 5 ' CTCCTACGGGAGGCAGCAG3 ', (Gentry, T.J., Wang, G., Rensing, C.and Pepper I.L. (2004) .Chlorobenzoate-degrading bacteria in similar pristine soilsexhibit different community structures and population dynamics in response to anthropogenic 2-, 3-, and4-chlorobenzoate levels.Microb.Ecology 48,90-102) with 1492R 5 ' GGTTACCTTGTTACGACTT3 ' (Wilson, K.H., Blitchington, R.B.and Green, R.C. (1990) Amplification of bacterial 16S ribosomal DNA with polymerase chainreaction.Journal of.Clinical Microbiology 28 1942-1946) goes out soil 16S rDNA and carries out electrophoresis detection for primer amplification;
(13) DNA that step (11) is extracted with EcoR I and Pst I makes double digestion and carries out electrophoresis detection.
Description of drawings
Fig. 1: be the electrophoresis detection figure that extracts the total DNA of same sample soil with 3 kinds of different methods.Numbering 1-3 is followed successively by the DNA that direct method, test kit method and the inventive method are extracted among the figure, and the point sample amount is 10ul.The molecular weight standard of DNA Marker I is followed successively by 15000bp from top to bottom, 10000bp, and 7500bp, 5000bp, 2500bp, 1000bp, (bp: base pair, sky, Beijing are Time Technology company limited to 250bp, catalog number (Cat.No.): MD115-01).
Fig. 2: be to extract the product electrophoresis detection figure that the total DNA of same sample soil does pcr amplification with 3 kinds of different methods.1-3 is followed successively by DNA that direct method, test kit method and the inventive method extract and does not dilute and do template, and 4-6 is followed successively by the DNA dilution that 3 kinds of methods extract and does template for 10 times, and the point sample amount is 10ul.The molecular weight standard of DNA Marker II is followed successively by 1500bp, 1000bp, 900bp, 800bp, 700bp from top to bottom, 600bp, 500bp, 400bp, 300bp, 200bp, (bp: base pair, sky, Beijing are Time Technology company limited to 100bp, catalog number (Cat.No.): MD109-01).
Fig. 3: the enzyme that is the DNA that extracts with the inventive method is cut product electrophoresis detection figure.1 DNA that cuts for enzyme not, 2 DNA that cut for enzyme, the point sample amount is respectively 5ul and 20ul (amount of DNA is identical).
Fig. 4: be the electrophoresis detection figure that extracts nature forest land, artificial forest land and paddy field soil DNA with the inventive method.1-3 is followed successively by the DNA of 3 kinds of soil samples, and the point sample amount is 10ul.
Good effect of the present invention such as table 1:
Table 1 the present invention and direct method and kit method and implementation result relatively
| Method | Advantage | Shortcoming |
| Direct method kit method the inventive method | Cracking condition is gentle, the dna fragmentation that obtains is relatively large, low price, do 1 pipe and need about 5 yuan simple, save time (about half an hour), convenient, the DNA purity of extraction is better, does not use that the poisonous organic solvents such as phenol, chloroform are simple, save time (about half an hour), convenient, DNA purity and the output extracted all are higher than conventional method and kit method, and low price is done 1 pipe and is no more than 3 yuan, does not use the poisonous organic solvents such as phenol, chloroform | Time-consuming, usually need more than 5 hours, the DNA that sometimes extracts is pure not, contain detritus acid and wait impurity, and when extracting, need use the poisonous organic solvents such as phenol, chloroform expensive, do 1 pipe and need 80 yuan, and purity is smaller not as the inventive method DNA, but can satisfy the requirement that PCR and enzyme are cut |
Embodiment
Embodiment 1: go into for extracting total DNA the soil (Orthic Anthrosols) from dry farming
Experiment soil is taken from Chinese Wuhan City, Hubei Province affiliated middle school of Hua Zhong Agriculture University playground top layer soil sample, and (this classification of soils is that dry farming goes into to be soil, pH=5.87, classification called after Orthic Anthrosols), the sieve in 2 millimeters * 2 millimeters apertures is crossed in the sampling back, and ℃ preservation is stand-by then-20.The concrete operations step is as follows:
(1) makes cracking tube: take by weighing each 10 gram of quartz sand and SiO 2 powder, earlier with rare nitric acid washing, then with deionized water wash and soaked overnight, again 80 ℃ of oven dry down, accurately take by weighing quartz sand and each 0.3 gram of SiO 2 powder of oven dry, pack into the centrifuge tube of 2ml, 121 ℃ of sterilizations 30 minutes;
(2) take by weighing soil sample 0.5 and restrain in the cracking tube of step (1), add the lysis buffer (preparation of lysis buffer: 0.1M Tris-HCl, 0.1M EDTA, 0.1MH
3PO
4Buffer, 1.5M NaCl, pH8.0,121 ℃ of sterilizations added CTAB after 30 minutes, making its final concentration is 1% (grams per milliliter)) mixing after 1 milliliter;
(3) with adhesive tape cracking tube is fixed on vortex mixed instrument (originate from its woods Bel instrument Manufacturing Co., Ltd of Haimen City, Jiangsu Province, product type is the QL-901 type) and goes up vibration 5 minutes;
(4) after under 10,000 * g centrifugal 1 minute supernatant liquor as much as possible is being gone in 1.5 milliliters of centrifuge tubes of a cleaning;
(5) 10, under 000 * g after centrifugal 2 minutes careful 0.4 milliliter of the supernatant liquor drawn in 2 milliliters of centrifuge tubes of a cleaning, with (the preparation of adsorbing base suspension: 0.3M HAc Buffer of adsorbing base suspension, 7M KSCN, about pH6, add with the washing of rare nitric acid and deionized water soaked overnight and at 80 ℃ of diatomite of drying down, making its final concentration is 5% (grams per milliliter) again) add 1 milliliter after shaking up;
(6) manually shake up 2 minutes gently, DNA is fully combined with adsorbing base;
(7) the careful suction abandoned 0.8 milliliter of supernatant liquor after under 10,000 * g centrifugal 10 seconds, all picks up (about 0.6 milliliter) after the resuspended matrix, moves on on the centrifugal purification post (the centrifugal purification post is bought from match Parkson, Shanghai gene engineering company limited) that contains collection tube;
(8) centrifugal 1 minute collecting precipitation under 10,000 * g removes the waste liquid in the collection tube;
(9) on the centrifugal purification post, add 0.5 milliliter of washings (preparation of washings: 12ml 3M NaAc mixes back adjust pH to 7.0 with 100ml ethanol), 10, centrifugal 1 minute washing matrix under 000 * g, remove the waste liquid in the collection tube, again under 10,000 * g centrifugal 2 minutes to remove residual washings;
(10) the centrifugal purification post is gone in 1.5 milliliters of centrifuge tubes of a cleaning, drying is 2 minutes under room temperature;
(11) in the middle of the centrifugal purification post, add the 100 microlitre TE damping fluid (preparations of TE damping fluid: 10mM Tris-HCl, 1mM EDTA, pH8.0, sterilized 30 minutes for 121 ℃) or deionized water, with 1 minute abundant wash-out of light finger bomb tube wall with assurance DNA, centrifugal 1 minute collection DNA under 10,000 * g;
For the parallel effect of comparison the present invention with existing direct method and test kit method, the applicant has carried out utilizing the experiment of direct method and test kit method simultaneously, utilize direct method from soil, to extract total DNA: to carry out (Zhou according to the Zhou reported method, J., Mary Ann Bruns, M.A., and Tiedje, M.J. (1996) DNA Recovery from Soils of Diverse Composition.Applied and Environmental Microbiology62 (2): 316-322); Utilize the test kit method to extract total DNA from soil: used kit is available from Q.BIOgene company, and the trade name of test kit is FastDNA SPIN Kit for Soil.Same the present invention of pedotheque that above-mentioned two kinds of methods are used, promptly sample preparation all uses the 0.5g soil sample to extract its DNA, then the DNA that extracts is dissolved in the TE damping fluid of 100 microlitres.
As shown in Figure 1, the total DNA result who adopts direct method, test kit method and method of the present invention to extract from soil has than big difference, total DNA output that preceding two kinds of methods are extracted is suitable, method of the present invention higher (the total DNA output that adopts spectrophotometer commonly used to detect is about 10ug) all exceeds 1 times than preceding two kinds of methods.
Embodiment 2: the DNA that embodiment 1 is extracted does the PCR detection
The DNA that extracts with present embodiment 1 is a template, with the primer of design: 338F 5 ' CTCCTACGGGAGGCAGCAG3 ' and 1492R5 ' GGTTACCTTGTTACGACTT3 ', amplification soil microorganisms 16S rDNA (16S ribosome rna gene).Utilize the total DNA of soil of indirect method, test kit method and method of the present invention preparation not dilute and dilute increase the respectively 16S rDNA of soil microorganisms of 10 times of templates of doing the PCR reaction.PCR reaction system following (50ul): ddH
2O:34.5ul; DNA:2ul; 10 * PCR buffer (TaKaRa): 5ul; 10 * MgCl
2(TaKaRa): 5ul; DNTP (Promega, every kind of 10mM): 1ul; 16S rDNA primer 338F (50ng/ul): 1ul; 16S rDNA primer 1492R (50ng/ul): 1ul; Taq Enzyme (TaKaRa, 5U/ul): 0.5ul.The PCR program is as follows: pre-sex change: 94 ℃ 5 minutes; Sex change: 94 ℃ 45 seconds; Annealing: 49 ℃ 45 seconds; Extend: 72 ℃ 1.5 minutes; Extend at last: 72 ℃ 10 minutes; Cycle number: 35.
The target DNA fragment size of amplification is 1151bp, and as shown in Figure 2, the 3rd to the 6th swimming lane has strong band between 1500bp and 1000bp, and the 1st and the 2nd swimming lane does not have band, and this DNA that shows that direct method and test kit method are extracted cannot directly do pcr amplification as template; Through can doing pcr amplification after 10 times of dilutions, and the DNA that method of the present invention is extracted does not dilute and dilutes 10 times and can satisfy the requirement of being PCR fully.
Embodiment 3: the DNA that uses the inventive method to extract is done enzyme cut detection
To the DNA that extracts with the inventive method among the embodiment 1 be EcoR I (G ↓ AATTC) and Pst I (double digestion of CTGCA ↓ G) (reference: J. Sa nurse Brooker, D.W. Russell. the molecular cloning experiment guide. the third edition. the .2003 of Science Press).Enzyme is cut system (20ul): ddH
2O:11ul; 10 * H buffer (TaKaRa): 2ul; EcoR I (TaKaRa, 15U/ul): 1ul; Pst I (TaKaRa, 15U/ul): 1ul; DNA:5ul.The enzyme tangent condition: 37 ℃ are spent the night.DNA after enzyme is cut as seen from Figure 3 presents a slice continuous strip-like, proves that DNA is cut into small segment, is the smear shape, illustrates that the DNA that extracts with the inventive method can satisfy the requirement that enzyme is cut.
Embodiment 4: use method of the present invention and extract the total DNA of soil from dissimilar pedotheques
Extract the DNA of 3 kinds of soil samples respectively according to the method for embodiment 1: the natural timbered soil (pH=6.65) and the artificial forest land of the Hua Zhong Agriculture University soil (pH=6.46) of Chinese Wuhan City, Hubei Province Hua Zhong Agriculture University Lion Rock, two kinds of soil are moistening Cambisol, classification called after Udic Cambisols; Near China's Hubei Province's Daye City's gold mine paddy field soil (pH=7.12), this soil are that water is ploughed into being soil, classification called after Stagnic Anthrosols.Electrophoresis detection figure is found out by Fig. 4 that as shown in Figure 43 kinds of soil samples in the present embodiment can both be prepared mass ratio DNA preferably, and its invention effect is as shown in table 2.
Table 2 is used the preparation effect of the present invention to the dissimilar total DNA of soil
| Soil type | The soil source | The output of DNA | The purity of DNA | The PCR situation that adapts to |
| Moistening Cambisol | The nature forest land | About 8ug | Higher | Adapt to |
| Moistening Cambisol | The artificial forest land | About 5ug | Higher | Adapt to |
| Water is ploughed into being soil | Paddy field soil | About 5ug | Higher | Adapt to |
Claims (4)
1, the small quality fast extraction method of the total DNA of a kind of soil, its key step comprises: specimen preparation, PCR and electrophoresis, concrete steps are as follows:
(1) makes cracking tube: take by weighing each 10 gram of quartz sand and SiO 2 powder, earlier with rare nitric acid washing, then with deionized water wash and soaked overnight, again 80 ℃ of oven dry down, accurately take by weighing quartz sand and each 0.3 gram of SiO 2 powder of oven dry, pack into the centrifuge tube of 2ml, 121 ℃ of sterilizations 30 minutes;
(2) take by weighing soil sample 0.5 and restrain in the cracking tube of step (1), add mixing behind 1 milliliter of the lysis buffer;
(3) with adhesive tape cracking tube is fixed on the vortex mixed instrument vibration 5 minutes;
(4) after under 10,000 * g centrifugal 1 minute supernatant liquor as much as possible is being gone in 1.5 milliliters of centrifuge tubes of a cleaning;
(5) at careful 0.4 milliliter of the supernatant liquor drawn after under 10,000 * g centrifugal 2 minutes in 2 milliliters of centrifuge tubes of a cleaning, add 1 milliliter after adsorbing base suspension shaken up;
(6) manually shake up 2 minutes gently, DNA is fully combined with adsorbing base;
(7) 0.8 milliliter of careful abandoning supernatant after under 10,000 * g centrifugal 10 seconds is all drawn the adsorbing base back that suspends again, moves on on the centrifugal purification post that contains collection tube;
(8) centrifugal 1 minute collecting precipitation under 10,000 * g removes the waste liquid in the collection tube;
(9) on the centrifugal purification post, add 0.5 milliliter of washings, under 10,000 * g centrifugal 1 minute the washing matrix, remove the waste liquid in the collection tube, again under 10,000 * g centrifugal 2 minutes to remove residual washings;
(10) the centrifugal purification post is gone in 1.5 milliliters of centrifuge tubes of a cleaning, drying is 2 minutes under room temperature;
(11) in the middle of the centrifugal purification post, add 100 microlitre TE damping fluid or deionized waters, with 1 minute abundant wash-out of light finger bomb tube wall with assurance DNA, centrifugal 1 minute collection DNA under 10,000 * g;
(12) DNA that extracts with step (11) is a template, is that primer amplification goes out soil 16S rDNA and carries out electrophoresis detection with 16S rDNA primer 338F 5 ' CTCCTACGGGAGGCAGCAG3 ' and 1492R5 ' GGTTACCTTGTTACGACTT3 ';
(13) DNA that step (11) is extracted with EcoR I and Pst I makes double digestion and carries out electrophoresis detection.
2, the described method of claim 1 is characterized in that, described lysis buffer prepares according to the following steps: 0.1M Tris-HCl, 0.1M EDTA, 0.1M H
3PO
4Buffer, 1.5M NaCl, pH8.0,121 ℃ of sterilizations added cetyl trimethylammonium bromide (CTAB) after 30 minutes, made its final concentration count 1% with grams per milliliter.
3, the described method of claim 1, it is characterized in that, described adsorbing base suspension preparation according to the following steps: 0.3M HAc Buffer, 7M KSCN, pH6.0,121 ℃ of sterilizations add after 30 minutes with rare nitric acid washing and deionized water soaked overnight and at 80 ℃ of diatomite that descend to dry, and make its final concentration count 5% with grams per milliliter.
4, the described method of claim 1 is characterized in that, described washings prepares according to the following steps: 12ml 3M NaAc is mixed the back transfer pH to 7.0 with 100ml ethanol.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101935647A (en) * | 2010-09-13 | 2011-01-05 | 原平皓(天津)生物技术有限公司 | Kit and method for extracting microbial DNA |
| CN102094004A (en) * | 2010-12-15 | 2011-06-15 | 南京师范大学 | Method for extracting ecological soil system matrix DNA |
| CN101210032B (en) * | 2006-12-26 | 2011-08-31 | 河南农业大学 | Extraction method for DNA in grape wine |
| CN101629174B (en) * | 2009-08-24 | 2011-11-23 | 吉首大学 | Simple, efficient and cheap method for purifying forest soil sample DNA |
| CN101709298B (en) * | 2009-12-14 | 2012-05-02 | 上海市农业科学院 | Soil DNA extracting method for evaluating diversity of microbial community of plant root system |
| CN104212794A (en) * | 2014-08-26 | 2014-12-17 | 沈阳农业大学 | Method for extracting plasmodiophora brassicae DNA of cruciferae vegetables and application of method |
| CN107083384A (en) * | 2017-06-12 | 2017-08-22 | 武汉科技大学 | Silicon column quick regeneration method |
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Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1456684A (en) * | 2003-03-26 | 2003-11-19 | 中国科学院生态环境研究中心 | Induction design plan for PCR-DGGE research on environment microorgan population |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101210032B (en) * | 2006-12-26 | 2011-08-31 | 河南农业大学 | Extraction method for DNA in grape wine |
| CN101629174B (en) * | 2009-08-24 | 2011-11-23 | 吉首大学 | Simple, efficient and cheap method for purifying forest soil sample DNA |
| CN101709298B (en) * | 2009-12-14 | 2012-05-02 | 上海市农业科学院 | Soil DNA extracting method for evaluating diversity of microbial community of plant root system |
| CN101935647A (en) * | 2010-09-13 | 2011-01-05 | 原平皓(天津)生物技术有限公司 | Kit and method for extracting microbial DNA |
| CN101935647B (en) * | 2010-09-13 | 2012-07-25 | 原平皓(天津)生物技术有限公司 | Kit and method for extracting microbial DNA |
| CN102094004A (en) * | 2010-12-15 | 2011-06-15 | 南京师范大学 | Method for extracting ecological soil system matrix DNA |
| CN104212794A (en) * | 2014-08-26 | 2014-12-17 | 沈阳农业大学 | Method for extracting plasmodiophora brassicae DNA of cruciferae vegetables and application of method |
| CN104212794B (en) * | 2014-08-26 | 2017-03-01 | 沈阳农业大学 | The extracting method of Plasmodiophora brassicae Causing Cruciferae Clubroot DNA and application |
| CN107083384A (en) * | 2017-06-12 | 2017-08-22 | 武汉科技大学 | Silicon column quick regeneration method |
| CN107083384B (en) * | 2017-06-12 | 2019-12-17 | 武汉科技大学 | Silicon column rapid regeneration method |
| US20200123528A1 (en) * | 2018-10-19 | 2020-04-23 | Winfield Solutions, Llc | Soil-based dna extraction |
| US11725202B2 (en) * | 2018-10-19 | 2023-08-15 | WinField Solutions. LLC | Soil-based DNA extraction |
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