CN101230342A - Soil sample total DNA extraction method for improving DNA quality - Google Patents
Soil sample total DNA extraction method for improving DNA quality Download PDFInfo
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Abstract
The invention relates to soil microbiology, in particular to a method of extracting the total DNA of the soil sample, which can improve the quality of DNA. The method includes the following steps: adding DNA extracting buffer to the soil sample, and then adding protease K; freeze thawing under -65 to 65 DEG C and centrifuging; discarding the precipitate; collecting the supernatant liquid; extracting and centrifuging by the same volume of the solution of phenol, chloroform and isoamylol, reserving the water phase; extracting one more time by the solution of chloroform and isoamylol; adding sodium acetate and cold isopropanol, and then centrifuging and collecting the DNA precipitate; washing by ethanol and drying in room temperature; adding TE buffer to dissolve, and gaining the DNA coarse extract; adding DNA extracting buffer in the coarse extract; and then adding the same volume of the solution of phenol, chloroform and isoamylol; repeating the preceding steps such as precipitating, washing and dissolving; finally, gaining the DNA of better quality. The method adopted by the invention can effectively remove the impurity in the DNA, which evidently increases the purity and quality of DNA.
Description
Technical field
The present invention relates to soil microbiology, is a kind of pedotheque total DNA extraction method of the DNA of raising quality specifically.
Background technology
Many researchs show that under existing state of the art, the most microorganisms (＞95%) in the natural ecological environment also can't successfully be cultivated in the laboratory.Because substratum can not satisfy the real site conditions of most of microbe, pure culture technigne just optionally allow those growth fractions rapidly, the fast microbe groups of condition of compatibility variation preferentially obtains growth and breeding.Can not represent real biological community structure composition in the environment by the microorganism that pure culture technigne obtains.
Over nearly 20 years, many molecular biology based on nucleic acid (or 16S rRNA gene sequence diversity), molecular ecology technique means are constantly developed and progressively ripe, for the structure of community that discloses microorganism in the ecosystem provides powerful technical support.These methods mainly contain: amplification rDNA restriction analysis technology (Amplified ribosomal DNA restriction analysis, ARDRA), single strand conformation polymorphism technology (Single strand conformation polymorphism, SSCP), (end) restrictive fragment length polymerphism technology ((Terminal) Restrictionfragment length polymorphism, T-RFLP), the denaturing gel electrophoresis technology (Denaturinggel electrophoresis, DGE) etc.All these research meanses based on microbe genome DNA in the environmental sample all need to extract earlier the DNA of capacity from sample, obtain a certain functional gene fragment by round pcr then, carry out deep analysis and research according to different research purposes again.
The DNA measurer that different DNA extraction method obtains has certain difference, analyzes operation but can satisfy that substantially follow-up PCR, clone, enzyme cut etc.Yet because the complicacy of environmental sample, the DNA quality that different DNA extraction methods is obtained but differs greatly.For the pedotheque of organic content than horn of plenty, traditional " mechanical breaking-wall method+zymetology effect " extracting method is limited in one's ability for the Impurity removals such as humic acid that contain in the soil.And remaining humic acid or some other impurity for pcr amplification, enzyme cut, the influence of operation such as clone is very big.
Summary of the invention
The object of the invention is to provide a kind of pedotheque total DNA extraction method of the DNA of raising quality.
For achieving the above object, the technical solution used in the present invention is:
Extracting method: 1) extract adding 1.0-5.5ml DNA extraction damping fluid in the sample, add Proteinase K and mixing that 0.3-1.0g quartz sand and 5-50 μ l concentration are again, place the vortex oscillation device to shake 10～20min to 0.5-5.0g; Then 30-40 ℃ water-bath 1-2 hour; Wherein the DNA extraction damping fluid is 50-150mM Tris-HCl, 50-100mM EDTA, and the 50-100mM phosphate buffered saline buffer, 0.5-2.5M NaCl, mass concentration is 0.5-2% cetyl trimethylammonium bromide (CTAB);
2) will shake in the afterreaction liquid and add sodium laurylsulfonate (SDS) the whirlpool concussion back multigelation 3 times that 200-2000 μ l mass concentration is 5-10%, then put into 65 ℃ of water-bath incubation 1-2h, and then the centrifugal 10-30min of 6000-10000 * g at ambient temperature, collect supernatant liquor, standby;
3) add isopyknic phenol-chloroform-primary isoamyl alcohol mixed solution in above-mentioned supernatant liquor, shake up to emulsus, the centrifugal 10-30min of 6000-10000 * g under the room temperature condition collects supernatant liquor; Then add isopyknic chloroform-primary isoamyl alcohol mixed solution in the supernatant liquor again, mixing is to emulsus, and the centrifugal 10-30min of 6000-10000 * g under the room temperature condition collects supernatant liquor;
4) with adding sodium-acetate and 0.8-1.2 times of volume cold isopropanol mixing of its 0.2-0.3 times volume in the above-mentioned supernatant liquor, place 1-2h at 4 ℃, the centrifugal 20-30min of 10000-12000 * g collects the DNA precipitation, and precipitation is through ethanol rinse 2-3 time, and after drying;
5) in dried DNA precipitation, add 50-100 μ L TE damping fluid and make its dissolving, obtain the DNA crude extract;
6) with the DNA extraction damping fluid of 500-1000 μ L in the above-mentioned coarse body fluid, repeating step 3)-5) can obtain the DNA extraction liquid of better quality.
Every 5-15min sample is spun upside down mixing in the described step 1) water-bath process; Described Proteinase K concentration is 10-20mg/L.Described through step 2) add DNA extraction damping fluid 100-600 μ l in the centrifugal gained precipitation, SDS solution 40-400 μ l, whirlpool concussion mixing, put into 65 ℃ of water-bath incubation 30-60min, the centrifugal 10-30min of 6000-10000 * g under the room temperature condition, collect supernatant liquor, its supernatant liquor and step 2) in the supernatant liquor of acquisition mix, standby.Described step 2) multigelation is 30-60min at-65 ℃ of freezing times for to be once-65 ℃ of freezing then melting 65 ℃ of speed simultaneously, and the thawing time is 10-20min in the time of 65 ℃.Described TE damping fluid is 5-20mM Tris-HCl, pH7.5-8.0, and 0.5-2mM EDTA, Tris-HCl pH is 7.5-8.0 in the described DNA extraction damping fluid, and EDTA pH is 7.5-8.0, and phosphate buffered saline buffer pH is 7.5-8.0.Phenol-chloroform in the described step 3)-primary isoamyl alcohol by volume mark ratio is 25: 24: 1, and chloroform-primary isoamyl alcohol by volume mark ratio is 24: 1.
The advantage that the present invention had:
1. DNA extraction method of the present invention is simple, does not need comparatively complex apparatus, and conventional biological chemistry or microbiology laboratory just can be finished; And the reagent that relates to is the reagent of routine analysis, molecular biology use, and all commercializations, can directly buy from reagent corresponding company.
2. the present invention is improving the DNA extraction method, improves the quality of the total DNA of soil that extracts.Make the electrophoretogram of DNA in the pedotheque more clear, and follow-up pcr amplification process become and is more prone to; The improvement of this method does not increase extra reagent, and the cost increase is also very little.
3. the DNA extraction method scope of application of the present invention is wider, except obtaining the soil of DNA than being easier to, black earth soil that organic content is higher or forest soil sample all can improve the soil DNA quality of extracting by this method, are suitable for subsequent P CR, the equimolecular biologic operation is cut, hybridizes, cloned to enzyme.
Description of drawings
(its electrophorogram is 0.8% sepharose to Fig. 1, is followed successively by from left to right: DNA marker for the soil DNA agarose gel electrophoresis synoptic diagram by the conventional DNA extraction method and the acquisition of improving one's methods; Swimming lane 2-4: the DNA product that ordinary method is extracted; 5-6: the DNA product that extract the back of improving one's methods).
Fig. 2 is that (wherein be followed successively by from left to right: swimming lane 1-3: the DNA product that extracts with ordinary method is the pcr amplification product that template is carried out to 16S rRNA gene fragment (the pcr amplification primer is 968f-gc/1401r) sepharose (1.5%) electrophorogram; DNA marker (swimming lane 4); Swimming lane 5-6: the DNA that extract the back of improving one's methods is the pcr amplification product that template is carried out).
Embodiment
Embodiment 1
1) accurately takes by weighing 0.5g northeast black earth pedotheque, place the sterilization centrifuge tube of 2mL, add 0.3g quartz sand, add again 800-1000 μ L DNA extraction damping fluid (100mM Tris-HCl[pH8.0], 100mM EDTA[pH8.0], 100mM phosphate buffered saline buffer (sodiumphosphate) [pH8.0], 1.5M NaCl is 1%CTAB) with 5 μ L Proteinase Ks (20mg/mL).Vortex concussion 10min, soil aggregate is fully scatter and with composition thorough mixing such as DNA extraction damping fluid, place 37 ℃ of water-bath temperature bath 1h, every 15min sample is overturn mixing several times in the water-bath process; Wherein phosphate buffered saline buffer is the 0.147g SODIUM PHOSPHATE, MONOBASIC; 5.08g seven hypophosphite monohydrate disodium hydrogens are dissolved in 200 ml waters and get final product.
2) will shaking afterreaction liquid, to add 200 μ L mass concentrations be 10%SDS solution, the whirlpool mixing, then multigelation is 3 times, and multigelation is for to be once-65 ℃ of freezing then melting 65 ℃ of speed, at-65 ℃ of freezing time 30min, the thawing time is 20min in the time of 65 ℃.Put into 65 ℃ of water-bath temperature then and bathe 2h, and shake gently once, make the abundant cracking of cell walls every 15-20min; And then the centrifugal 10min of 6000 * g at ambient temperature, collect supernatant liquor in new sterilization centrifuge tube.
Wherein add 160 μ L DNA extraction damping fluids in the gained precipitation, 40 μ L 10%SDS solution, concussion, dissolution precipitation thing again, and placing 65 ℃ of water-bath 40-50min, the centrifugal 10min of 6000 * g collects supernatant, it is mixed with above-mentioned centrifugal gained supernatant liquor, standby.
3) will add isopyknic phenol in the above-mentioned mixing supernatant: chloroform: primary isoamyl alcohol (volume ratio is 25: 24: 1), shake up solution to emulsus, the centrifugal 10min of 6000 * g draws the upper strata water; And add isopyknic chloroform at the upper strata aqueous phase: primary isoamyl alcohol (volume ratio is 24: 1) shakes up the centrifugal 10min of 6000 * g.Draw upper strata water (look the experiment particular case and can repeat this step more once);
4) will add 3mol/L sodium-acetate (pH5.2) and 0.8 times of volume cold isopropanol of 0.2 times of volume in the above-mentioned supernatant liquor, mixing is placed 1-2h for 4 ℃ gently, the centrifugal 20-30min of 12000 * g abandons supernatant, collects the DNA precipitation, cold washing with alcohol with 100 μ L 70% precipitates drying at room temperature 2-3 time.
5) (1mMEDTA) the dissolving DNA precipitation obtains the DNA crude extract for 10mM Tris-HCl, pH8.0 to add 60 μ L TE damping fluids in the dry postprecipitation;
6) the DNA extraction damping fluid of adding 500 μ L in the DNA crude extract, then repeating step 3)-5), finally can obtain high-quality DNA crude extract (referring to Fig. 1).
As shown in Figure 1, swimming lane 2-4DNA electrophoretogram has conditions of streaking, and the DNA collection of illustrative plates of swimming lane 5-6 is clear, do not have hangover.The application of sample amount of considering each swimming lane DNA is identical, and visible conventional DNA extraction method can not more effectively be removed some contained in DNA crude extract impurity, and improved DNA extraction method then can remedy this deficiency.
Embodiment 2
1) accurately takes by weighing 1.0g Changbai Mountain Korean pine forest pedotheque, place the sterilization centrifuge tube of 5mL, add 0.5g quartz sand, add again 1500 μ L DNA extraction damping fluids (100mM Tris-HCl[pH7.5], 100mM sodium EDTA[pH7.5], 100mM phosphate buffered saline buffer (sodiumphosphate) [pH7.5], 1.5M NaCl is 1%CTAB) with 15 μ L Proteinase Ks (20mg/mL).Vortex concussion 20min, soil aggregate is fully scatter and with composition thorough mixing such as DNA extraction damping fluid, place 37 ℃ of water-bath temperature bath 1.5h, every 10min sample is overturn mixing several times in the water-bath process; Wherein phosphate buffered saline buffer is that 0.735g SODIUM PHOSPHATE, MONOBASIC and 25.4g seven hypophosphite monohydrate disodium hydrogens are dissolved in 1000 ml waters and get final product.
2) will shake afterreaction liquid and add 400 μ L 10%SDS solution, the whirlpool mixing, then multigelation is 3 times, and multigelation is for to be once-65 ℃ of freezing then melting 65 ℃ of speed, and at-65 ℃ of freezing time 40min, the thawing time is 10min in the time of 65 ℃.。Put into 65 ℃ of water-bath temperature then and bathe 2h, and shake gently once, make the abundant cracking of cell walls every 15-20min; And then the centrifugal 20min of 10000 * g at ambient temperature, collect supernatant liquor in new sterilization centrifuge tube.
Wherein add 350 μ L DNA extraction damping fluids in the gained precipitation, 100 μ L 10%SDS solution, concussion, dissolution precipitation thing again, and placing 65 ℃ of water-bath 30-40min, the centrifugal 20min of 10000 * g collects supernatant, it is mixed with above-mentioned centrifugal gained supernatant liquor, standby.
3) will add isopyknic phenol in the above-mentioned mixing supernatant: chloroform: primary isoamyl alcohol (volume ratio is 25: 24: 1), shake up solution to emulsus, the centrifugal 20min of 10000 * g draws the upper strata water; And add isopyknic chloroform at the upper strata aqueous phase: primary isoamyl alcohol (volume ratio is 24: 1) shakes up the centrifugal 20min of 10000 * g.Draw upper strata water (look the experiment particular case and can repeat this step more once);
4) will add 3mol/L sodium-acetate (pH 5.2) and 1.0 times of volume cold isopropanols of about 0.3 times of volume in the above-mentioned supernatant liquor, mixing is placed 1-2h for 4 ℃ gently, the centrifugal 20min of 12000 * g abandons supernatant, collects the DNA precipitation, cold washing with alcohol with 100 μ L70% precipitates drying at room temperature 2-3 time.
5) (1mMEDTA) the dissolving DNA precipitation obtains the DNA crude extract for 10mM Tris-HCl, pH7.5 to add 100 μ L TE damping fluids in the dry postprecipitation;
6) the DNA extraction damping fluid of adding 600 μ L in the DNA crude extract, then repeating step 3)-5), finally can obtain high-quality DNA crude extract (referring to Fig. 2).
Under identical PCR condition, the DNA that extracts with ordinary method is that template does not obtain goal gene product (swimming lane 1-3) known to Fig. 2, and after improving one's methods, DNA purity is improved, and successfully amplifies 16S rRNA gene segment (shown in swimming lane 5-6).
Embodiment 3
Difference from Example 1 is:
1) accurately takes by weighing the 5g pedotheque, add 1g quartz sand, add again 5mL DNA extraction damping fluid (50mM Tris-HCl[pH7.5], 80mM sodium EDTA[pH7.5], 60mM phosphate buffered saline buffer (sodium phosphate) [pH7.5], 2.5M NaCl is 2%CTAB) with 50 μ L Proteinase Ks (20mg/mL).Vortex concussion 15min, soil aggregate is fully scatter and with composition thorough mixing such as DNA extraction damping fluid, place 30 ℃ of water-bath temperature bath 2h, every 5min sample is overturn mixing several times in the water-bath process;
2) will shake afterreaction liquid and add 2000 μ L 10%SDS solution, the centrifugal 30min of 8000 * g collects supernatant liquor in new sterilization centrifuge tube at ambient temperature.
Wherein add 600 μ L DNA extraction damping fluids in the gained precipitation, 40 μ L 10%SDS solution, concussion, dissolution precipitation thing again, and placing 65 ℃ of water-bath 50-60min, the centrifugal 30min of 8000 * g collects supernatant, it is mixed with above-mentioned centrifugal gained supernatant liquor, standby.
3) will add isopyknic phenol in the above-mentioned mixing supernatant: chloroform: primary isoamyl alcohol (volume ratio is 25: 24: 1), shake up solution to emulsus, the centrifugal 20min of 10000 * g draws the upper strata water; And add isopyknic chloroform at the upper strata aqueous phase: primary isoamyl alcohol (volume ratio is 24: 1) shakes up the centrifugal 30min of 8000 * g.
4) will add 3mol/L sodium-acetate (pH5.2) and 1.2 times of volume cold isopropanols of 0.2 times of volume in the above-mentioned supernatant liquor, mixing is placed 1-2h for 4 ℃ gently, the centrifugal 25min of 11000 * g abandons supernatant, collects the DNA precipitation, cold washing with alcohol with 100 μ L70% precipitates drying at room temperature 2-3 time.
5) (1mMEDTA) the dissolving DNA precipitation obtains the DNA crude extract for 10mM Tris-HCl, pH7.5 to add 80 μ L TE damping fluids in the dry postprecipitation;
6) the DNA extraction damping fluid of adding 1000 μ L in the DNA crude extract, then repeating step 3)-5), finally can obtain high-quality DNA crude extract.
Embodiment 4
Difference from Example 1 is:
1) accurately takes by weighing the 3g pedotheque, add 0.8g quartz sand, add again 3mL DNA extraction damping fluid (80mM Tris-HCl[pH7.5], 50mM sodium EDTA[pH7.5], 50mM phosphate buffered saline buffer (sodium phosphate) [pH7.5], 0.5M NaCl is 0.5%CTAB) with 30 μ L Proteinase Ks (20mg/mL).
2) will shake afterreaction liquid and add 1000 μ L 10%SDS solution.
Wherein add 100 μ L DNA extraction damping fluids in the gained precipitation, 400 μ L 10%SDS solution, concussion, dissolution precipitation thing again, and placing 65 ℃ of water-bath 40-50min, the centrifugal 15min of 9000 * g collects supernatant, it is mixed with above-mentioned centrifugal gained supernatant liquor, standby.
3) the centrifugal 15min of 9000 * g.
5) (2mMEDTA) the dissolving DNA precipitation obtains the DNA crude extract for 20mM Tris-HCl, pH8.0 to add 80 μ L TE damping fluids in the dry postprecipitation;
6) the DNA extraction damping fluid of adding 800 μ L in the DNA crude extract, then repeating step 3)-5), finally can obtain high-quality DNA crude extract.
Embodiment 5
Difference from Example 1 is:
1) accurately takes by weighing the 2g pedotheque, add 1g quartz sand, add 1mL DNA extraction damping fluid and 20 μ L Proteinase Ks (20mg/mL) again.
2) will shake afterreaction liquid and add 800 μ L 10%SDS solution.
5) (2mMEDTA) the dissolving DNA precipitation obtains the DNA crude extract for 20mM Tris-HCl, pH8.0 to add 50 μ L TE damping fluids in the dry postprecipitation;
6) the DNA extraction damping fluid of adding 900 μ L in the DNA crude extract, then repeating step 3)-5), finally can obtain high-quality DNA crude extract.
The add-on of above solution all can be according to the corresponding expansion of extracting sample, and corresponding increasing.
Claims (6)
1. pedotheque total DNA extraction method that improves the DNA quality is characterized in that:
1) extracts adding 1.0-5.5ml DNA extraction damping fluid in the sample to 0.5-5.0g, add Proteinase K and mixing that 0.3-1.0g quartz sand and 5-50 μ l concentration are again, place the vortex oscillation device to shake 10～20min; Then 30-40 ℃ water-bath 1-2 hour; Wherein the DNA extraction damping fluid is 50-150mM Tris-HCl, 50-100mM EDTA, and the 50-100mM phosphate buffered saline buffer, 0.5-2.5MNaCl, mass concentration is the 0.5-2% cetyl trimethylammonium bromide;
2) will shake in the afterreaction liquid and add the sodium laurylsulfonate whirlpool concussion back multigelation 3 times that 200-2000 μ l mass concentration is 5-10%, then put into 65 ℃ of water-bath incubation 1-2h, and then the centrifugal 10-30min of 6000-10000 * g at ambient temperature, collect supernatant liquor, standby;
3) add isopyknic phenol-chloroform-primary isoamyl alcohol mixed solution in above-mentioned supernatant liquor, shake up to emulsus, the centrifugal 10-30min of 6000-10000 * g under the room temperature condition collects supernatant liquor; Then add isopyknic chloroform-primary isoamyl alcohol mixed solution in the supernatant liquor again, mixing is to emulsus, and the centrifugal 10-30min of 6000-10000 * g under the room temperature condition collects supernatant liquor;
4) with adding sodium-acetate and 0.8-1.2 times of volume cold isopropanol mixing of its 0.2-0.3 times volume in the above-mentioned supernatant liquor, place 1-2h at 4 ℃, the centrifugal 20-30min of 10000-12000 * g collects the DNA precipitation, and precipitation is through ethanol rinse 2-3 time, and after drying;
5) in dried DNA precipitation, add 50-100 μ L TE damping fluid and make its dissolving, obtain the DNA crude extract;
6) with the DNA extraction damping fluid of 500-1000 μ L in the above-mentioned coarse body fluid, repeating step 3)-5) can obtain the DNA extraction liquid of better quality.
2. by the pedotheque total DNA extraction method of the described raising of claim 1 DNA quality, it is characterized in that: every 5-15min sample is spun upside down mixing in the described step 1) water-bath process; Described Proteinase K concentration is 10-20mg/L.
3. by the described pedotheque total DNA extraction method that improves the DNA quality of claim 1, it is characterized in that: described through step 2) add DNA extraction damping fluid 100-600 μ l in the centrifugal gained precipitation, SDS solution 40-400 μ l, whirlpool concussion mixing, put into 65 ℃ of water-bath incubation 30-60min, the centrifugal 10-30min of 6000-10000 * g under the room temperature condition collects supernatant liquor, its supernatant liquor and step 2) in the supernatant liquor that obtains mix, standby.
4. by the described pedotheque total DNA extraction method that improves the DNA quality of claim 1, it is characterized in that: described step 2) multigelation is for to be once-65 ℃ of freezing then melting 65 ℃ of speed, be 30-60min at-65 ℃ of freezing times simultaneously, the thawing time is 10-20min in the time of 65 ℃.
5. by the described pedotheque total DNA extraction method that improves the DNA quality of claim 1, it is characterized in that: described TE damping fluid is 5-20mM Tris-HCl, pH7.5-8.0,0.5-2mM EDTA, Tris-HCl pH is 7.5-8.0 in the described DNA extraction damping fluid, EDTA pH is 7.5-8.0, and phosphate buffered saline buffer pH is 7.5-8.0.
6. by the pedotheque total DNA extraction method of the described raising of claim 1 DNA quality, it is characterized in that: phenol-chloroform in the described step 3)-primary isoamyl alcohol by volume mark ratio is 25: 24: 1, and chloroform-primary isoamyl alcohol by volume mark ratio is 24: 1.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101974513A (en) * | 2010-11-18 | 2011-02-16 | 福建农林大学 | Extraction method of soil microbe genome DNA and total RNA |
| CN102260666A (en) * | 2010-05-24 | 2011-11-30 | 中国水产科学研究院东海水产研究所 | Method for extracting high-purity total DNA from pond bottom mud sample |
| CN102418151A (en) * | 2011-09-19 | 2012-04-18 | 福建农林大学 | Method for constructing cDNA (complementary DNA) library of soil microbe |
| 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 |
| CN103045586A (en) * | 2013-01-18 | 2013-04-17 | 赵树民 | Method for extracting infant saliva internal genome DNA applied to whole genome sequencing |
| CN104911178A (en) * | 2015-06-19 | 2015-09-16 | 厦门大学 | Method for simultaneously extracting microbial intracellular and extracellular DNAs (deoxyribonucleic acids) in sewage biological treatment water sample |
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| 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 |
| CN102260666A (en) * | 2010-05-24 | 2011-11-30 | 中国水产科学研究院东海水产研究所 | Method for extracting high-purity total DNA from pond bottom mud sample |
| CN101974513A (en) * | 2010-11-18 | 2011-02-16 | 福建农林大学 | Extraction method of soil microbe genome DNA and total RNA |
| CN101974513B (en) * | 2010-11-18 | 2012-05-23 | 福建农林大学 | Extraction method of soil microbe genome DNA and total RNA |
| CN102418151A (en) * | 2011-09-19 | 2012-04-18 | 福建农林大学 | Method for constructing cDNA (complementary DNA) library of soil microbe |
| CN102418151B (en) * | 2011-09-19 | 2013-04-17 | 福建农林大学 | Method for constructing cDNA (complementary DNA) library of soil microbe |
| CN103045586A (en) * | 2013-01-18 | 2013-04-17 | 赵树民 | Method for extracting infant saliva internal genome DNA applied to whole genome sequencing |
| CN104911178A (en) * | 2015-06-19 | 2015-09-16 | 厦门大学 | Method for simultaneously extracting microbial intracellular and extracellular DNAs (deoxyribonucleic acids) in sewage biological treatment water sample |
| CN110295162A (en) * | 2019-06-17 | 2019-10-01 | 广东省生态环境技术研究所 | A kind of DNA extraction reagent and extracting method for Fe-mn Nodules of Soils microorganism |
| CN110295162B (en) * | 2019-06-17 | 2022-03-29 | 广东省科学院生态环境与土壤研究所 | DNA extraction reagent and extraction method for soil iron-manganese nodule microorganisms |
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