CN102643797B - Method for extracting total DNAs of soil microorganisms at high purity - Google Patents
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Abstract
The invention discloses a method for extracting total DNAs of soil microorganisms at high purity. CTAB (Cetyltrimethyl Ammonium Bromide), lysozyme, protease and SDS (Sodium Dodecyl Sulfonate) act together to perform lysis of cells and simultaneously freeze and thaw the cells repeatedly; therefore, an excellent cell lysis effect is achieved; the humic acid is removed by utilizing PVP (Polyvinyl Pyrrolidone), CaC12 and BSA (Bull Serum Albumin); and the DNAs are precipitated by utilizing isopropanol so that the purity of the obtained DNAs is high. The method is simple and convenient; and without being purified, the DNAs obtained by the method can be used for subsequent PCR (Polymerase Chain Reaction) analysis and DGGE (Denaturing Gradient Gel Electrophoresis) analysis. The purity of the extracted DNAs meets the requirement for directly performing subsequent molecular biological research; the extracted DNA segments can be subjected to 16SrDNA amplification and DGGE atlas analysis; glue cutting recovery and basic group sequencing can be performed on specific stripes of the DGGE band; unknown bacteria can be identified through comparison with the conventional sequences in an RDP (Ribosomal Database Project) database or by establishing a new sequence probe; and therefore, the structural composition of the microbial community can be determined, and a foundation can be laid for researching the structural diversity and ecological functions of the soil microorganisms in the rhizosphere of the mulberry in future.
Description
Technical field
The present invention relates to Molecular Ecology Techniques field, be specifically related to a kind of extracting method of Soil of Mulberry Garden microorganism total DNA.
Background technology
For a long time, the Study on Diversity of relevant soil microorganisms realizes by separation and Culture.Yet most microbial populations still can not be realized pure culture, this becomes a restrictive factor of diversity of soil microorganism research.Along with deepening continuously of structure of soil microbial community research, by the research method of modern molecular biology technique, avoided the defect that traditional research method can not Overall Acquisition diversity of soil microorganism information as PCR-RFLP, PCR-SSCP, PCR-DGGE etc., the diversity that can show by soil microbial DNA, reflect soil microbial population structure situation truly, and efficient, the high-quality extraction of soil microbial community genome DNA is the precondition of structure of soil microbial community being studied from molecular biology level.Due to soil physical chemistry complicated component, often containing humic acid, phenolic compound, heavy metal ion etc. affects the material of DNA molecular operation, pedotheque processing and experiment condition are more complicated, length consuming time, so that obtaining high purity microbe genome DNA tool from edatope acquires a certain degree of difficulty, therefore DNA extraction technological innovation receives much concern.Reported at present multiple soil microorganisms total DNA extraction method.From the method for soil extract microorganism total DNA, be roughly divided into two classes: direct extraction method and indirect DNA extraction method.The DNA purity that indirect method is extracted is high, but its shortcoming is the bacterium obtaining, only accounts for 25%～50% of total flora, and the DNA that direct method is extracted surpasses 60% of bacteria total DNA.Direct method owing to can extract more full DNA of bacteria, laborsaving, DNA output is high, thereby development is very fast.Tsai etc. are used SDS and the total DNA of lysozyme lysis cell extraction, Tiedje etc. are used PVP (polyvinylpyrrolidone) and CTAB (cetyl trimethylammonium bromide) to remove humic acid, effect is fine, the people such as Bourrain adopt the method from active sludge, to extract DNA, and the people such as Reddy have extracted DNA from compost.
The generation of the formation of the diversity of Soil of Mulberry Garden microorganism and vital movement and Soil of Mulberry Garden structure and improvement, Evolution of Soil Fertility, mulberry tree root disease, the supply of mulberry tree nutritive substance and vine growth and development etc. are closely related, but have not yet to see the report that culture method research Soil of Mulberry Garden microbial diversity is exempted from application.
Summary of the invention
The present invention is the molecular ecology experimental technique system based on setting up mulberry tree rhizosphere structure of soil microbial community, use for reference the extracting method of the total DNA of existing soil microorganisms, a kind of Soil of Mulberry Garden microorganism total DNA extracting method that can be directly used in efficiently pcr analysis is provided.
For achieving the above object, the invention discloses following technical scheme:
One. material is selected
For examination soil on March 7th, 2011 pick up from Crop Institute, Hunan Academy of Agricultural Sciences's production mulberry field (114 ° 19 of east longitude ', 30 ° 29 of north latitude ', height above sea level 29m) mulberry tree rhizosphere, mulberry tree breed is "Hur" mulberry No. 32.Collection soil is yellow clay, and sampling depth is 5～10cm topsoil, by 5 method samplings, after mixing, puts into sterile bag, and 4 ℃ of preservations, complete the extraction of soil microorganisms genome DNA in 24h.
Two. reagent is selected
In the main agents that the present invention adopts: cetyl trimethylammonium bromide (CTAB), sodium laurylsulfonate (SDS), PVP K30 (PVP K30), PEG 8000 (PEG8000), urea, deionized formamide are all purchased from magnificent biotechnology company limited; N,O-Diacetylmuramidase, proteolytic enzyme are all purchased from Sigma company; Silver Nitrate is purchased from the 95 Ling Jiu factory of the Chinese People's Liberation Army; λ DNA/HindIII digest DNA Marker, 2000DL DNA Marker and Taq HS are all purchased from Dalian precious biotechnology company limited; 16SrDNA universal primer is synthetic by Shanghai Ying Jun biotechnology company limited; Soil extract test kit
soil DNA Kit (50) is purchased from Omega company.
Three. the total DNA By Direct Pyrolysis of soil microorganisms method
Method one CTAB-proteolytic enzyme-SDS-multigelation method
Get 5g soil sample, add wherein 13.5mL DNA extraction liquid I (Tris-HCl 100mmol/L, EDTA 100mmol/L, Na
3pO
4100mmol/L, NaCl 1.5mol/L, 1%CTAB; PH 8.0) and a little quartz sand, vortex oscillation device vibration 3min; Add again 20mg/mL Proteinase K 15 μ L, mix, incubation 1h in 37 ℃, 230r/min shaking table; Add 1.5mL 10%SDS, 65 ℃ of water-bath 1h; Multigelation 3 times, the centrifugal 10min of 8500r/min; Get supernatant liquor, add isopyknic phenol/chloroform/primary isoamyl alcohol (volume ratio 25: 24: 1) extracting, the centrifugal 10min of 8500r/min; Get supernatant liquor, add isopyknic chloroform/primary isoamyl alcohol (volume ratio 24: 1) extracting again, the centrifugal 10min of 8500r/min, gets supernatant liquor, standby.
Method two PVP pre-treatment-CTAB-N,O-Diacetylmuramidase-proteolytic enzyme-SDS-multigelation method
Getting 5g soil sample adds 20g/L sodium-metaphosphate damping fluid (containing 10g/L PVP-K30; PH 8.5) 30mL, shaking table vibration 15min, the centrifugal 5min of 8500r/min, gets precipitation, repeated washing 3 times; Get precipitation, add 13.5mL DNA extraction liquid I (Tris-HCl 100mmol/L, EDTA 100mmol/L, Na
3pO
4100mmol/L, NaCl 1.5mol/L, 1%CTAB; PH 8.0) and a little quartz sand, vortex oscillation device vibration 3min; Add 100mg/mL N,O-Diacetylmuramidase 150 μ L, mix, incubation 1h in 37 ℃, 230r/min shaking table; Add again 20mg/mL Proteinase K 15 μ L, mix, 37 ℃ of water-bath 1h; Add 1.5mL 10%SDS, 65 ℃ of water-bath 1h; Multigelation 3 times, the centrifugal 10min of 8500r/min; Get supernatant liquor, add isopyknic phenol/chloroform/primary isoamyl alcohol (volume ratio 25: 24: 1) extracting, the centrifugal 10min of 8500r/min; Get supernatant liquor, add isopyknic chloroform/primary isoamyl alcohol (volume ratio 24: 1) extracting again, the centrifugal 10min of 8500r/min, gets supernatant liquor, standby.
Method three PVP pre-treatment-CTAB, CaCl
2, BSA-N,O-Diacetylmuramidase-proteolytic enzyme-SDS-multigelation method
Getting 5g soil sample adds 20g/L sodium-metaphosphate damping fluid (containing 10g/L PVP-K30; PH 8.5) 30mL, shaking table vibration 15min, the centrifugal 5min of 8500r/min, gets precipitation, repeated washing 3 times; Get precipitation, add 13.5mL DNA extraction liquid II (Tris-HCl 100mmol/L, EDTA 100mmol/L, Na
3pO
4100mmol/L, NaCl 1.5mol/L, 1%CTAB, 2%CaCl
2, 1 μ g/mL BSA; PH 8.0) and a little quartz sand, vortex oscillation device vibration 3min.Subsequent operations is identical with method two.
Method four CTAB, PVP, CaCl
2, BSA-N,O-Diacetylmuramidase, proteolytic enzyme-SDS-multigelation method
Get 5g soil sample, add 13.5mL DNA extraction liquid III (Tris-HCl 100mmol/L, EDTA 100mmol/L, Na
3pO
4100mmol/L, NaCl 1.5mol/L, 1%CTAB, 2%CaCl
2, 1 μ g/mL BSA, 2%PVP; PH 8.0) and a little quartz sand, vortex oscillation device concussion 3min; Add 150 μ L 100mg/mL N,O-Diacetylmuramidases, 15 μ L 20mg/mL Proteinase Ks, mix, incubation 1h in 37 ℃, 230r/min shaking table.Subsequent operations is identical with method two.
Method five CTAB, CaCl
2, BSA-N,O-Diacetylmuramidase, proteolytic enzyme-SDS, PVP-multigelation method
Get 5g loading, add 13.5mL DNA extraction liquid II (Tris-HCl 100mmol/L, EDTA 100mmol/L, Na
3pO
4100mmol/L, NaCl 1.5mol/L, 1%CTAB, 2%CaCl
2, 1 μ g/mL BSA; PH 8.0) and a little quartz sand, vortex oscillation device vibration 3min; Add 100mg/mL N,O-Diacetylmuramidase 150 μ L, 20mg/mL Proteinase K 15 μ L, mix, incubation 1h in 37 ℃, 230r/min shaking table; Add 1.5mL 10%SDS and 0.3g PVP, mix, 65 ℃ of water-bath 1h; Multigelation 3 times.Subsequent operations is identical with method two.
Method six test kit methods
Adopt the soil extract test kit of Omega company
soil DNA Kit (50) extracts.
Four. the total DNA intermediate processing of soil microorganisms
Method one isopropanol precipitating method
In supernatant liquor, add 0.7 times of volume Virahol and 0.1 times of volume NaAc ,-20 ℃ of placements are spent the night, 8500r/min, 4 ℃ of centrifugal 20min, precipitation 70% washing with alcohol, the centrifugal 10min of 12500r/min, precipitation room temperature is dried, and adds 200 μ LTE dissolution precipitations ,-20 ℃ of preservations.
The method two PEG8000 precipitator method
In supernatant liquor, add 0.5 times of volume 25%PEG 8000 and 0.1 times of volume 5mol/LNaCl, 4 ℃ of placements are spent the night, 8500r/min, 4 ℃ of centrifugal 20min, precipitation 70% washing with alcohol, the centrifugal 10min of 12500r/min, precipitation room temperature is dried, and adds 200 μ L TE dissolution precipitations ,-20 ℃ of preservations.
Five. the quality examination of the total DNA of soil microorganisms
The purity of 1.DNA and detection by quantitative are measured D (230nm), D (260nm), D (280nm) value of DNA solution with ultraviolet spectrophotometer, according to multiple * 50 of formula dsDNA=D (260nm) * dilution, calculate the mass concentration (ng/ μ L) of DNA, converse the DNA amount that every gram of soil extracts, and according to D (260nm)/D (230nm), D (260nm)/D (280nm), detect the purity of DNA.With 1% agarose gel electrophoresis, detect and analyze simultaneously.
2.16SrDNAV3 fragment PCR be take the Soil of Mulberry Garden microorganism total DNA that extracts and is directly carried out PCR as template.Forward and reverse primer is respectively: GC-F341 (5 '-CGCCCGCCG CGC GCG GCG GGC GGG GCG GGGGCACGGGGG G CCT ACG GGA GGC AGC AG-3 ') and R518 (5 '-ATTACC GCG GCT GCTGG-3 ').Reaction system: 10 * PCR buffer (containing Mg2+), 3 μ L, 2.5mmol/L dNTPs 2.4 μ L, each 0.6 μ L of 10 μ mol/L upstream and downstream primers, Taq enzyme 1U, template DNA 30ng, finally adds ddH2O to 30 μ L.PCR program: 94 ℃ of denaturation 5min; 94 ℃ of sex change 1min, annealing 1min, 72 ℃ are extended 1min, 36 circulations (annealing temperature is from 65 ℃～55 ℃, and each circulation reduces by 0.5 ℃, and 15 circulations subsequently keep 55 ℃); 72 ℃ of final 7min that extend.PCR product detects by 2% agarose gel electrophoresis.In triplicate.
3. the separated PCR product of the separated 16SrDNA V3 fragment PCR products application JY-TD331 denaturing gradient gel electrophoresis instrument (Jun Yi east, Beijing electrophoresis equipment company limited) of denaturing gradient gel electrophoresis.Get 15 μ L PCR products, add 10 μ L6 * loading buffer to carry out denaturing gradient gel electrophoresis (DGGE), deposition condition: 8% polyacrylamide, electrophoretic buffer is 1 * TAE, 50%～80% denaturing agent (urea and deionized formamide), temperature 60 C, voltage 100V, electrophoresis time 17h.Electrophoresis finishes rear silver and dyes colour developing.
Six. interpretation of result
1. the preliminary screening of mulberry tree rhizosphere soil microorganisms total DNA extraction method
Adopt 5 kinds of different lysis methods and 2 kinds of DNA intermediate processings to combine, thereby obtain the method for 8 kinds of different total DNA of direct extraction mulberry tree rhizosphere soil microorganisms, and to take the total DNA of mulberry tree rhizosphere soil microorganisms that test kit method extracts be reference, utilize its D of determined by ultraviolet spectrophotometry (230nm), D (260nm), D (280nm) value.With purity and the quality of D (260nm)/D (280nm) representation DNA, the degree of polluting with humic acid in D (260nm)/D (230nm) representation DNA.Table 1 data presentation the effect of Different Extraction Method.
Method 2, with CTAB-proteolytic enzyme-SDS-multigelation lysing cell, obtain DNA productive rate higher, but humic acid is seriously polluted; Method 3,4,5,6 all adopts 2% sodium-metaphosphate damping fluid (containing 1%PVP-K30 on the basis of method 2; PH8.5) pre-wash fully discharges microorganism from soil, and to improving DNA productive rate, but method 3,6 does not reach ideal effect, and method 4,5 effects are better, and has played certain effect to removing humic acid.Wherein, method 4 and method 5 further adopt BSA, CaCl on the basis of method 2
2remove humic acid, the purity of DNA is increased, the pollution level of humic acid decreases.Method 8,9 has been saved PVP pre-treatment, and PVP adds in cleavage step thereafter, but impact little.The DNA purity that test kit extracts is high, and humic acid pollution level is low, and still, DNA productive rate is low, and test kit is expensive.
As shown in Figure 1, the molecular mass of the DNA that different methods extracts, more than 23kb, is all genomic dna, and DNA do not interrupted, and is large fragment DNA, can be used for follow-up molecular biological analysis.Consider DNA purity and quality, humic acid pollution level and productive rate, method 4,5,8,9, in conjunction with physics, chemistry, biological 3 kinds of method lysing cell, is added PVP, BSA, CaCl in DNA extraction liquid
2further remove humic acid, save loaded down with trivial details pre-wash step, the DNA productive rate obtaining is higher than 11.2 μ g/g, D (260nm)/D (230nm) is greater than 2.11, D (260nm)/D (280nm) is greater than 1.71, has all reached and directly carry out the requirement of PCR in purity and humic acid pollution level.
The comparison of several mulberry tree rhizosphere of table 1 soil microorganisms total DNA extraction method
The not purified mulberry tree rhizosphere soil microorganisms genome DNA that the different methods of take extracts is template, bacterial 16 S rDNA V3 district directly increases, thereby judge and in DNA extraction sample, whether have inhibitor and be enough to suppress Taq enzymic activity, causing it can not obtain amplified production.Amplified production by 1.5% agarose gel electrophoresis detected result as Fig. 2.Employing method 4,5,8 and the 9 total DNA that extract can successfully amplify 16SrDNA V3 fragment, and the template DNA that employing method 1,2,3,6,7 is extracted fails to amplify object band, illustrate that DNA profiling is impure, contain certain impurity, suppressed follow-up PCR reaction.
By the direct loading of product after method 4,5,8 and 9 total DNA cloning of extracting, the diversity of the Soil of Mulberry Garden microflora by denaturing gradient gel electrophoresis detection display, thereby further verify that this test sets up the feasibility of mulberry tree rhizosphere soil microorganisms total DNA extraction method, result as shown in Figure 3.Total DNA of 4 kinds of method extractions all can obtain than more rich bacterial flora diversity collection of illustrative plates, but the Phylogenetic diversity of bacteria that method 8,9 is extracted is obviously wanted the height of ratio method 4,5.Hence one can see that, extracts highly purified DNA profiling very crucial to pcr amplification.
The present invention has following beneficial effect:
1. for the singularity of Soil of Mulberry Garden, when utilizing CTAB, N,O-Diacetylmuramidase, proteolytic enzyme and SDS acting in conjunction with lysing cell to its multigelation smudge cells, determine CTAB, N,O-Diacetylmuramidase, proteolytic enzyme, SDS concentration that applicable Soil of Mulberry Garden is processed, obtained good lysis effect.Determine the reaction conditions that is suitable for Soil of Mulberry Garden, improved DNA productive rate.Method therefor is simple and convenient, does not need purified can be used for follow-up pcr analysis and DGGE analysis.
2. for the singularity of Soil of Mulberry Garden, this law has been saved tediously long, loaded down with trivial details DNA washing step, saves time and avoids DNA loss, and directly add CaCl in DNA extraction liquid
2, BSA and PVP, determined the reaction conditions of applicable Soil of Mulberry Garden, make easy and simple to handlely, humic acid removal effect is obvious, this is innovation of the present invention.In addition, CTAB not only can be used for lysis, also assists in removing humic acid.The isopropanol precipitating reaction system that is suitable for Soil of Mulberry Garden of establishing makes the DNA purity that the present invention extracts improve 6.3%-8.4% than existing extraction effect best technique, reaches the needs that directly carry out subsequent molecular.
3. the DNA fragmentation that utilizes the inventive method to extract can carry out amplification and the DGGE atlas analysis of 16SrDNA, and can cut glue recovery, base order-checking to the specific band of DGGE bands of a spectrum, compare with existing sequence in RDP database, or identify unknown bacterium by setting up new sequence probe, thereby determine that biological community structure forms, and lays the foundation for studying from now on mulberry tree rhizosphere its community diversity in different habitats and ecological functions.
Accompanying drawing explanation
Fig. 1 different methods extracts the electrophoretogram (M. λ DNA/HindIII molecule marker 1～9 different methods extracts total DNA, and swimming lane numbering is consistent with table 1 numbering) of the total DNA of mulberry tree rhizosphere soil microorganisms
Fig. 2 different methods extracts the 16S rDNA pcr amplification result (M.DL2000DNA molecule marker 1～9 different methods extracts the 16S rDNA amplification of total DNA, and swimming lane numbering is consistent with table 1 numbering) of the total DNA of mulberry tree rhizosphere soil microorganisms
Fig. 3 different methods extracts the DGGE collection of illustrative plates (swimming lane numbering is consistent with table 1 numbering) of the 16S rDNA of the total DNA of mulberry tree rhizosphere soil microorganisms
Embodiment
For simple and object clearly, below the present invention is described further in conjunction with the embodiments:
Embodiment 1:
PVP pre-treatment-CTAB-N,O-Diacetylmuramidase-proteolytic enzyme-SDS-multigelation method.Getting 5g soil sample adds 20g/L sodium-metaphosphate damping fluid (containing 10g/LPVP-K30, pH 8.5) 30mL, shaking table vibration 15min, the centrifugal 5min of 8500r/min, gets precipitation, repeated washing 3 times, get precipitation, add 13.5mL DNA extraction liquid I and a little quartz sand, vortex oscillation device vibration 3min, add 100mg/mL N,O-Diacetylmuramidase 150 μ L, mix, incubation 1h in 37 ℃, 230r/min shaking table, add again 20mg/mL Proteinase K 15 μ L, mix, 37 ℃ of water-bath 1h, add 1.5mL 10%SDS, 65 ℃ of water-bath 1h, multigelation 3 times, the centrifugal 10min of 8500r/min, get supernatant liquor, add isopyknic phenol/chloroform/primary isoamyl alcohol (volume ratio 25: 24: 1) extracting, the centrifugal 10min of 8500r/min, get supernatant liquor, add isopyknic chloroform/primary isoamyl alcohol (volume ratio 24: 1) extracting again, the centrifugal 10min of 8500r/min, get supernatant liquor, add isopyknic chloroform/primary isoamyl alcohol (volume ratio 24: 1) extracting again, the centrifugal 10min of 8500r/min, get supernatant liquor, in supernatant liquor, add 0.7 times of volume Virahol and 0.1 times of volume NaAc,-20 ℃ of placements are spent the night, 8500r/min, 4 ℃ of centrifugal 20min, precipitation 70% washing with alcohol, the centrifugal 10min of 12500r/min, precipitation room temperature is dried, add 200 μ L TE dissolution precipitations,-20 ℃ of preservations obtain Soil of Mulberry Garden microorganism total DNA.
Embodiment 2:
PVP pre-treatment-CTAB, CaCl2, BSA-N,O-Diacetylmuramidase-proteolytic enzyme-SDS-multigelation method are got 5g soil sample and are added 20g/L sodium-metaphosphate damping fluid (containing 10g/LPVP-K30; PH 8.5) 30mL, shaking table vibration 15min, the centrifugal 5min of 8500r/min, gets precipitation, repeated washing 3 times; Get precipitation, add 13.5mL DNA extraction liquid II and a little quartz sand, vortex oscillation device vibration 3min; Add 100mg/mL N,O-Diacetylmuramidase 150 μ L, mix, incubation 1h in 37 ℃, 230r/min shaking table; Add again 20mg/mL Proteinase K 15 μ L, mix, 37 ℃ of water-bath 1h; Add 1.5mL 10%SDS, 65 ℃ of water-bath 1h; Multigelation 3 times, the centrifugal 10min of 8500r/min; Get supernatant liquor, add isopyknic phenol/chloroform/primary isoamyl alcohol (volume ratio 25: 24: 1) extracting, the centrifugal 10min of 8500r/min; Get supernatant liquor, add isopyknic chloroform/primary isoamyl alcohol (volume ratio 24: 1) extracting again, the centrifugal 10min of 8500r/min, get supernatant liquor, in supernatant liquor, add 0.7 times of volume Virahol and 0.1 times of volume NaAc ,-20 ℃ of placements are spent the night, 8500r/min, 4 ℃ of centrifugal 20min, precipitation is used 70% washing with alcohol, the centrifugal 10min of 12500r/min, and precipitation room temperature is dried, add 200 μ LTE dissolution precipitations ,-20 ℃ of preservations.Obtain Soil of Mulberry Garden microorganism total DNA.
Embodiment 3:
CTAB, PVP, CaCl2, BSA-N,O-Diacetylmuramidase, proteolytic enzyme-SDS-multigelation method.Get 5g soil sample, add 13.5mLDNA extracting solution III and a little quartz sand, vortex oscillation device concussion 3min; Add 150 μ L 100mg/mL N,O-Diacetylmuramidases, 15 μ L20mg/mL Proteinase Ks, mix, incubation 1h in 37 ℃, 230r/min shaking table; Add again 20mg/mL Proteinase K 15 μ L, mix, 37 ℃ of water-bath 1h; Add 1.5mL 10%SDS, 65 ℃ of water-bath 1h; Multigelation 3 times, the centrifugal 10min of 8500r/min; Get supernatant liquor, add isopyknic phenol/chloroform/primary isoamyl alcohol (volume ratio 25: 24: 1) extracting, the centrifugal 10min of 8500r/min; Get supernatant liquor, add isopyknic chloroform/primary isoamyl alcohol (volume ratio 24: 1) extracting again, the centrifugal 10min of 8500r/min, get supernatant liquor, add 0.7 times of volume Virahol and 0.1 times of volume NaAc in supernatant liquor ,-20 ℃ of placements are spent the night, 8500r/min, 4 ℃ of centrifugal 20min, precipitation 70% washing with alcohol, the centrifugal 10min of 12500r/min, precipitation room temperature is dried, add 200 μ L TE dissolution precipitations ,-20 ℃ of preservations, obtain Soil of Mulberry Garden microorganism total DNA.
Embodiment 4:
CTAB, CaCl2, BSA-N,O-Diacetylmuramidase, proteolytic enzyme-SDS, PVP-multigelation method.Get 5g soil sample, add 13.5mLDNA extracting solution II and a little quartz sand, vortex oscillation device vibration 3min; Add 100mg/mL N,O-Diacetylmuramidase 150 μ L, 20mg/mL Proteinase K 15 μ L, mix, incubation 1h in 37 ℃, 230r/min shaking table; Add 1.5mL 10%SDS and 0.3g PVP, mix, 65 ℃ of water-bath 1h; Multigelation 3 times; The centrifugal 10min of 8500r/min; Get supernatant liquor, add isopyknic phenol/chloroform/primary isoamyl alcohol (volume ratio 25: 24: 1) extracting, the centrifugal 10min of 8500r/min; Get supernatant liquor, add isopyknic chloroform/primary isoamyl alcohol (volume ratio 24: 1) extracting again, the centrifugal 10min of 8500r/min, get supernatant liquor, add 0.7 times of volume Virahol and 0.1 times of volume NaAc in supernatant liquor ,-20 ℃ of placements are spent the night, 8500r/min, 4 ℃ of centrifugal 20min, precipitation 70% washing with alcohol, the centrifugal 10min of 12500r/min, precipitation room temperature is dried, add 200 μ LTE dissolution precipitations ,-20 ℃ of preservations, obtain Soil of Mulberry Garden microorganism total DNA.
Embodiment 5:
CTAB, PVP, CaCl2, BSA-N,O-Diacetylmuramidase, proteolytic enzyme-SDS-multigelation method.Get 5g soil sample, add 13.5mLDNA extracting solution III and a little quartz sand, vortex oscillation device concussion 3min; Add 150 μ L 100mg/mL N,O-Diacetylmuramidases, 15 μ L20mg/mL Proteinase Ks, mix, incubation 1h in 37 ℃, 230r/min shaking table; Add again 20mg/mL Proteinase K 15 μ L, mix, 37 ℃ of water-bath 1h; Add 1.5mL 10%SDS, 65 ℃ of water-bath 1h; Multigelation 3 times, the centrifugal 10min of 8500r/min; Get supernatant liquor, add isopyknic phenol/chloroform/primary isoamyl alcohol (volume ratio 25: 24: 1) extracting, the centrifugal 10min of 8500r/min; Get supernatant liquor, add isopyknic chloroform/primary isoamyl alcohol (volume ratio 24: 1) extracting again, the centrifugal 10min of 8500r/min, get supernatant liquor, in supernatant liquor, add 0.5 times of volume 25%PEG 8000 and 0.1 times of volume 5mol/L NaCl, 4 ℃ of placements are spent the night, 8500r/min, 4 ℃ of centrifugal 20min, precipitation is used 70% washing with alcohol, the centrifugal 10min of 12500r/min, and precipitation room temperature is dried, add 200 μ L TE dissolution precipitations ,-20 ℃ of preservations.Obtain Soil of Mulberry Garden microorganism total DNA.
Claims (2)
1. high purity is extracted a method of the total DNA of soil microorganisms, it is characterized in that, comprises the steps:
Adopt CTAB, CaCl
2, BSA-N,O-Diacetylmuramidase, proteolytic enzyme-SDS, PVP-multigelation method; Get 5g mulberry tree rhizosphere soil sample, add 13.5mL DNA extraction liquid II and a little quartz sand, vortex oscillation device vibration 3min; Add 100mg/mL N,O-Diacetylmuramidase 150 μ L, 20mg/mL Proteinase K 15 μ L, mix, incubation 1h in 37 ℃, 230r/min shaking table; Add 1.5mL10%SDS and 0.3g PVP, mix, 65 ℃ of water-bath 1h; Multigelation 3 times; The centrifugal 10min of 8500r/min; Get supernatant liquor, add the extracting of isopyknic phenol/chloroform/primary isoamyl alcohol, phenol/chloroform/primary isoamyl alcohol volume ratio 25: 24: 1, the centrifugal 10min of 8500r/min; Get supernatant liquor, add the extracting again of isopyknic chloroform/primary isoamyl alcohol, chloroform/primary isoamyl alcohol volume ratio 24: 1, the centrifugal 10min of 8500r/min, gets supernatant liquor; In supernatant liquor, add 0.7 times of volume Virahol and 0.1 times of volume NaAc,-20 ℃ of placements are spent the night, 8500r/min, 4 ℃ of centrifugal 20min, precipitation 70% washing with alcohol, the centrifugal 10min of 12500r/min, precipitation room temperature is dried, add 200 μ L TE dissolution precipitations ,-20 ℃ of preservations, obtain the total DNA of mulberry tree rhizosphere soil microorganisms;
Described DNA extraction liquid II composition is Tris-HC1100mmol/L, EDTA100mmol/L, Na
3pO
4100mmol/L, NaC11.5mol/L, 1%CTAB, 2%CaCl
2, 1 μ g/mL BSA; PH8.0; Described soil sample, picks up from mulberry tree rhizosphere soil, and the degree of depth is 5～10cm topsoil, by 5 method samplings, puts into sterile bag, 4 ℃ of preservations after mixing.
2. high purity as claimed in claim 1 is extracted the purposes of the method for the total DNA of soil microorganisms, it is characterized in that: the method is being carried out amplification and the DGGE atlas analysis of 16SrDNA, compare with existing sequence in RDP database, or identify unknown bacterium by setting up new sequence probe, thereby determine the purposes of biological community structure composition, structure diversity and ecological functions thereof.
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| CN103789300B (en) * | 2014-02-26 | 2015-08-12 | 济南大学 | A kind of extracting method of epoxy propane saponified wastewater active sludge macro genome DNA |
| CN103820434A (en) * | 2014-03-19 | 2014-05-28 | 四川省农业科学院土壤肥料研究所 | Method for extracting total DNA (deoxyribonucleic acid) of fungal hyphae |
| CN104178481A (en) * | 2014-09-05 | 2014-12-03 | 福建师范大学 | Kit for extracting soil microbial genome DNA within 30 minute |
| 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 |
| CN110628873A (en) * | 2019-09-25 | 2019-12-31 | 北京交通大学 | Method for removing extracellular nucleic acid of pretreated sludge |
| CN110628694B (en) * | 2019-11-12 | 2022-05-27 | 山东省农作物种质资源中心 | Method for extracting pea hypocotyl mitochondria |
| CN111518798A (en) * | 2020-06-02 | 2020-08-11 | 苏州路易兴环保科技有限公司 | Chinese herbal medicine microorganism culture method and DNA extraction method |
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