CN101974513A - Extraction method of soil microbe genome DNA and total RNA - Google Patents
Extraction method of soil microbe genome DNA and total RNA Download PDFInfo
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Abstract
The invention provides a method for effectively extracting crop rhizosphere soil microbe genome DNA and total RNA, which comprises the following steps: firstly, removing humus and other impurities which severely disturb nucleic acid extraction in soil samples by aluminum sulfate; further crushing soil microbe cells by glass beads; adding sodium dodecyl sulfonate (SDS) and LiCl solutions for cracking cells and dissociating nucleic acids; adding the mixture of phenol, chloroform and isoamylol for extracting; and then, depositing the extraction solution by isopropanol and sodium acetate to finally acquire the soil microbe metagenome DNA and total RNA. The method is suitable for extraction of the DNA and total RNA of soil in different regions. The invention establishes an extraction technology which has the advantages of simple and easy operation processes and higher purity of the prepared sample, and can provide an important basis for the research of soil metagenomics.
Description
Technical field
The present invention relates to the extracting method of a kind of soil microbe genome DNA and total RNA, belong to biological technical field.
Technical background
Because the restriction of factors such as traditional microbiological research method, development and utilization as the basic Microbial resources of life science source and biotechnology innovation once once had been absorbed in the deadlock that high frequency repeats to screen, and the continuous development of modern biotechnology has overcome these difficult problems gradually.In the research of modern soil microecology, directly extract microbe genome DNA in the soil, and at the advanced at present Protocols in Molecular Biology of this basis utilization, amplified fragment length polymorphism (AFLP) technology for example, end limit fragment length polymorphism (T-RFLP) technology, single strand conformation poly morphism (SSCP), sex change/temperature gradient gel elec-trophoresis (TGGE) (DGGE/TGGE) technology, overcome the drawback that soil microorganisms information that traditional cultural method caused is lost in a large number to a certain extent, more comprehensive, accurately disclose rhizosphere soil microorganism structure of community and function diversity.
But for simple cultured microorganism, soil microbial DNA extracts and has some difficulties, and the DNA that ordinary method obtains is difficult to satisfy the downstream molecules operational requirement.This mainly is to be caused by following two kinds of reasons:
(1) soil constituent complexity comprises the inhibition of multiple PCR, for example: heavy metal, polyose, Polyphenols, soil ulmin etc.Wherein soil ulmin is owing to have the similar character with DNA, therefore can be in the conventional extracting of DNA and the DNA co-precipitation, and the existence of soil ulmin minute quantity just can suppress PCR consumingly and react.
(2) environmental microorganism such as soil more is difficult to cracking with respect to simple cultured microorganism.It is low the DNA extraction method of conventional pure culture microorganism to be used for the microorganism cells cleavage rate that environmental microorganism DNA extraction such as soil obtained, and therefore the DNA that obtains can not reflect the extraordinary abundance of primary sample.In addition, also cause DNA output on the low side.
Therefore need development be applicable to convenient, fast, the practical method that soil microbial DNA extracts, solve and use DNA sample that ordinary method obtained to have PCR inhibition such as soil ulmin and the lysis rate is low, DNA yields poorly problem.
In addition, effectively extract total RNA of soil microorganisms, can provide important assurance for the gene expression pattern of analyzing soil microorganisms under the varying environment condition, thereby enrich the research contents of grand genomics.Yet in the process of extracting soil RNA, except existing the identical difficulty with the extraction soil DNA, ubiquitous RNA enzyme constitutes greatly threat to the stable existence of RNA, and this also is that soil RNA extracts the few reason of product in the present domestic and international market.In view of the above, develop the method for a kind of high efficiency extraction soil genomic dna and RNA, seem very necessary for carrying out the grand genomics research of soil on a large scale.
Summary of the invention:
The invention provides the method for a kind of effective extraction crop rhizosphere soils microbe genome DNA and total RNA, the nucleic acid purity of utilizing this method to extract is higher, and output is higher.
The extracting method of a kind of soil microbe genome DNA of the present invention and total RNA comprises the removal of pedotheque impurity, soil microorganisms lysis, the free precipitation that reaches of nucleic acid, and it is characterized in that: concrete steps are as follows:
(1) removal of pedotheque impurity: the fresh crop rhizosphere soils sample that takes by weighing 0.2～1.0g adds 40～200 μ l 1M Tris-HCl (pH=5.5), V in the aseptic centrifuge tube of 2ml
1μ l aseptic double-distilled water (V
1=900 μ l-V
2) and V
2μ l 0.2M Al
2(SO
4)
3The aqueous solution, wherein V
2=60～300 μ l(are owing to the content difference of impurity such as different regions or different planting material rhizosphere soil soil ulmin, available gradient V
2=60,90,120,150,180,210,240,270,300 pairs supply examination soil to try to carry), vortex concussion 30s; Add 1/3 V then
2Volume 4M NaOH solution, and V
3Volume 0.1M Tris-HCl (pH=8) (V
3=1300-4/3 V
2-V
1), vortex concussion 30s, with pH value 〉=8 of the 4M NaOH solution adjusting soil solution, the centrifugal 2min of 3500rpm removes supernatant and calculates supernatant volume V with liquid-transfering gun
4
(2) soil microorganisms lysis: in the centrifugal precipitation that obtains of step (1), add V
5The 0.1M Tris-HCl of volume (pH 8) (V
5=V
4-650 μ l), add 0.5～0.8g 0.5mm granulated glass sphere again, 0.3～0.5g 0.1mm granulated glass sphere and 1～2 4mm granulated glass sphere, and then add the 325 μ l 10%SDS aqueous solution (pH=8), 325 μ l EB solution (pH=8), vortex concussion 30s, ice bath 1min, and then vortex concussion 1min, ice bath 5min, vortex concussion 1min once more, 4 ℃ of centrifugal 1min of 11000rpm then, it is standby to get supernatant.
Wherein EB (pH=8) solution is formulated as: take by weighing 2.416g LiCl, 1.211g Tris, 3.506g EDTA, add the distilled water dissolving and regulate pH=8.0 with 4M NaOH solution, be settled to 100ml.
(3) the free and precipitation of nucleic acid: get supernatant that 600～800 μ l steps (2) obtain to the aseptic centrifuge tube of 1.5ml, add and the isopyknic phenol of supernatant: chloroform: primary isoamyl alcohol (volume ratio=25:24:1), the vortex concussion, ice bath 5min, per minute concussion during this time 1 time, last 4 ℃ of centrifugal 15min of 16000rpm.Supernatant after centrifugal is transferred to 1.5ml centrifuge tube (centrifuge tube steeps the back autoclaving with the 0.1%DEPC water logging), adds and the isopyknic chloroform of supernatant: primary isoamyl alcohol (volume ratio=24:1), the vortex concussion, 4 ℃ of centrifugal 15min of 16000rpm get supernatant.Supernatant is transferred to new 1.5ml centrifuge tube (0.1%DEPC water logging bubble back autoclaving), adds and the isopyknic chloroform of supernatant: primary isoamyl alcohol (volume ratio=24:1), the vortex concussion, 4 ℃ of centrifugal 15min of 16000rpm get supernatant.Supernatant is transferred to another 1.5ml centrifuge tube (0.1%DEPC water logging bubble back autoclaving), the Virahol, the 0.1 times of supernatant volume 3M sodium-acetate (pH=5.5) that add 0.7 times of supernatant volume, 4 ℃ leave standstill 3h or-20 ℃ of precipitations 10min, 4 ℃ of centrifugal 60min of 18000rpm then.Remove supernatant, precipitation 5ml 75% ethanol (preparation of 0.1%DEPC water) washed twice, 4 ℃ of wash conditions, the centrifugal 5min of 18000rpm.Remove supernatant, be deposited in and dry up in the aseptic technique platform and with 30 μ l 0.1%DEPC water dissolution, obtain containing DNA and the total solution of RNA ,-80 ℃ of preservations.
The extracting method of described soil microbe genome DNA and total RNA, preferred step is as follows:
(1) removal of pedotheque impurity: the fresh crop rhizosphere soils sample that takes by weighing 0.5g adds Tris-HCl, the V of 100 μ l 1M pH=5.5 in the aseptic centrifuge tube of 2ml
1μ l aseptic double-distilled water and V
2μ l 0.2M Al
2(SO
4)
3The aqueous solution, wherein V
1=900 μ l-V
2, V
2=60～300 μ l, vortex concussion 30s; Add 1/3 V then
2Volume 4M NaOH solution, and V
3The Tris-HCl of volume 0.1M pH=8, wherein V
3=1300-4/3 V
2-V
1, vortex concussion 30s, with pH value 〉=8 of the 4M NaOH solution adjusting soil solution, the centrifugal 2min of 3500rpm removes supernatant and calculates supernatant volume V with liquid-transfering gun
4
(2) soil microorganisms lysis: in the centrifugal precipitation that obtains of step (1), add V
5The Tris-HCl of the 0.1M pH=8 of volume, wherein V
5=V
4-650 μ l, add 0.5g 0.5mm granulated glass sphere again, 0.3g 0.1mm granulated glass sphere and 1 4mm granulated glass sphere, and then add the SDS aqueous solution of 325 μ l, 10% pH=8, the EB solution of 325 μ l pH=8, vortex concussion 30s, ice bath 1min, and then vortex concussion 1min, ice bath 5min, vortex concussion 1min once more, 4 ℃ of centrifugal 1min of 11000rpm then, it is standby to get supernatant;
Being formulated as of EB solution: take by weighing 2.416g LiCl, 1.211g Tris, 3.506g EDTA, add the distilled water dissolving and regulate pH=8.0, be settled to 100ml with 4M NaOH solution.
(3) the free and precipitation of nucleic acid: get supernatant that 750 μ l steps (2) obtain to the aseptic centrifuge tube of 1.5ml, add and the isopyknic phenol of supernatant, chloroform and primary isoamyl alcohol mixed solution, phenol wherein: chloroform: primary isoamyl alcohol volume ratio=25:24:1, the vortex concussion, ice bath 5min, per minute concussion during this time 1 time, last 4 ℃ of centrifugal 15min of 16000rpm; Supernatant after centrifugal is transferred to the 1.5ml centrifuge tube, adds and the isopyknic chloroform of supernatant and primary isoamyl alcohol mixed solution, wherein chloroform: primary isoamyl alcohol volume ratio=24:1, the vortex concussion, 4 ℃ of centrifugal 15min of 16000rpm get supernatant; Supernatant is transferred to new 1.5ml centrifuge tube, adds and the isopyknic chloroform of supernatant and primary isoamyl alcohol mixed solution, wherein chloroform: primary isoamyl alcohol volume ratio=24:1, the vortex concussion, 4 ℃ of centrifugal 15min of 16000rpm get supernatant; Supernatant is transferred to another 1.5ml centrifuge tube, adds the Virahol of 0.7 times of supernatant volume and the sodium-acetate of 0.1 times of supernatant volume 3M pH=5.5,4 ℃ leave standstill 3h or-20 ℃ of precipitations 10min, 4 ℃ of centrifugal 60min of 18000rpm then; Remove supernatant, precipitation 5ml 75% ethanol (with the preparation of 0.1%DEPC water) washed twice under 4 ℃ of temperature, the centrifugal 5min of 18000rpm then; Remove supernatant, be deposited in and dry up in the aseptic technique platform and with 30 μ l 0.1%DEPC water dissolution, obtain containing DNA and the total solution of RNA ,-80 ℃ of preservations.Above-mentioned 1.5ml centrifuge tube is earlier with 0.1%DEPC water logging bubble back autoclaving.
Extracting method of the present invention extracts pedotheque earlier with serious interfere RNA such as Tai-Ace S 150 removal soil ulmin impurity, further use the broken soil microorganisms cell of granulated glass sphere, add SDS and LiCl solution lysing cell, free nucleic acid again, after adding phenol, chloroform and iso pentane alcohol mixture extraction, extraction liquid finally obtains soil microorganisms macro genome DNA and total RNA with Virahol and sodium-acetate precipitation.The purity of the nucleic acid of gained can reach 1.724, and concentration reaches 12.2 μ g/ μ l.After the DNA that extracts and RNA carried out purifying respectively, the DNA of purifying is carried out the amplification of 16srDNA, total RNA of purifying is carried out sxemiquantitative PCR and the quantitative fluorescent PCR analysis of reverse transcription and 16srRNA, the results are shown in Figure 1, confirm the validity of method extraction soil DNA of the present invention and total RNA.Repetition test proves, soil genomic dna and RNA that this method is extracted, and integrity is good, and purity is higher, can be used for genomic library construction, RT-PCR, the northern-blot etc. in downstream after being further purified.
Remarkable advantage of the present invention:
1. the soil that is suitable for different areas, different sources has the broad-spectrum suitability.
2. the DNA of gained and RNA integrity are good, purity is higher.
3. extract convenient, as to need not complexity treatment process, the condition of extraction is not strict.
Description of drawings
Fig. 1 is rhizosphere soil DNA and the isolating collection of illustrative plates of RNA;
Fig. 2 is the different V of sugarcane soil
2Volume Al
2(SO
4)
3The extraction result; Wherein swimming lane 1,2, and 3,4,5,6,7 represent 120,150,180,210,240,270 and 300 μ l respectively;
Fig. 3 is the electrophoresis detection result that Different Crop rhizosphere soil DNA and RNA extract; The included part of solid box is DNA; Frame of broken lines be RNA; A wherein: tobacco, B: Root of Heterophylly Faalsestarwort, C: sugarcane, D: paddy rice, E: glutinous rehmannia.
Specific embodiment
Embodiment 1
The extracting method of soil microbe genome DNA of the present invention and total RNA, concrete steps are as follows:
(1) raw material: the fresh cane rhizosphere soil, from Foochow, Fujian (University Of Agriculture and Forestry In Fujian teaching farm);
(2) removal of pedotheque impurity: the fresh cane rhizosphere soil sample that takes by weighing 0.5g adds 100 μ l 1M Tris-HCl (pH=5.5), V in the aseptic centrifuge tube of 2ml
1μ l aseptic double-distilled water (V
1=900 μ l-V
2) and V
2μ l 0.2M Al
2(SO
4)
3The aqueous solution (V
2Gradient gets 60,90, and 120,150,180,210,240,270,300 μ l are to trying respectively to carry for examination soil), vortex concussion 30s; Add 1/3 V then
2Volume 4M NaOH solution, and V
3Volume 0.1M Tris-HCl (pH=8) (V
3=1300-4/3 V
2-V
1), vortex concussion 30s, with pH value=8 of the 4M NaOH solution adjusting soil solution, the centrifugal 2min of 3500rpm removes supernatant and calculates supernatant volume V with liquid-transfering gun
4
(3) soil microorganisms lysis: in the centrifugal precipitation that obtains of step (1), add V
5The 0.1M Tris-HCl of volume (pH 8) (V
5=V
4-650 μ l), add 0.5g 0.5mm granulated glass sphere again, 0.3g 0.1mm granulated glass sphere and 1 4mm granulated glass sphere, and then add the 325 μ l 10%SDS aqueous solution (pH=8), 325 μ l EB solution (pH=8), vortex concussion 30s, ice bath 1min, and then vortex concussion 1min, ice bath 5min, vortex concussion 1min once more, 4 ℃ of centrifugal 1min of 11000rpm then, it is standby to get supernatant.
Wherein EB (pH=8) solution is formulated as: take by weighing 2.416g LiCl, 1.211g Tris, 3.506g EDTA, add the distilled water dissolving and regulate pH=8.0 with 4M NaOH solution, be settled to 100ml.
(3) the free and precipitation of nucleic acid: get supernatant that 750 μ l steps (2) obtain to the aseptic centrifuge tube of 1.5ml, add 750 μ l phenol: chloroform: primary isoamyl alcohol (volume ratio=25:24:1), vortex concussion, ice bath 5min, per minute concussion during this time 1 time, last 4 ℃ of centrifugal 15min of 16000rpm.Supernatant after centrifugal is transferred to 1.5ml centrifuge tube (centrifuge tube steeps the back autoclaving with the 0.1%DEPC water logging), adds and the isopyknic chloroform of supernatant: primary isoamyl alcohol (volume ratio=24:1), the vortex concussion, 4 ℃ of centrifugal 15min of 16000rpm get supernatant.Supernatant is transferred to new 1.5ml centrifuge tube (0.1%DEPC water logging bubble back autoclaving), adds and the isopyknic chloroform of supernatant: primary isoamyl alcohol (volume ratio=24:1), the vortex concussion, 4 ℃ of centrifugal 15min of 16000rpm get supernatant.Supernatant is transferred to another 1.5ml centrifuge tube (0.1%DEPC water logging bubble back autoclaving), the Virahol, the 0.1 times of supernatant volume 3M sodium-acetate (pH=5.5) that add 0.7 times of supernatant volume, 4 ℃ leave standstill 3h or-20 ℃ of precipitations 10min, 4 ℃ of centrifugal 60min of 18000rpm then.Remove supernatant, precipitation 5ml 75% ethanol (preparation of 0.1%DEPC water) washed twice, 4 ℃ of wash conditions, the centrifugal 5min of 18000rpm.Remove supernatant, be deposited in and dry up in the aseptic technique platform and with 30 μ l 0.1%DEPC water dissolution, obtain containing the solution of DNA and total RNA, get the solution of 5 water-soluble DNA of containing of μ l 0.1%DEPC and RNA, electrophoresis detection in 1% sepharose, detected result is seen Fig. 2, DNA by more visible the 2nd swimming lane and the band of RNA are the most clear, band brightness is the most obvious, and the impurity background in the swimming lane is light, illustrates that this adds this volume Al
2(SO
4)
3Extraction effect the best, promptly extract the suitableeest Al of fresh cane rhizosphere soil
2(SO
4)
3Volume is 150 μ l, adds the Al of 150 μ l
2(SO
4)
3Can farthest remove the soil ulmin in the soil, guarantee that again the loss of DNA and RNA is minimum simultaneously.
Embodiment 2
(1) removal of pedotheque impurity:
Take by weighing fresh cane (Foochow, Fujian), glutinous rehmannia (Jiaozhuo, Henan), Root of Heterophylly Faalsestarwort (Zherong, Fujian), the tobacco (Kunming, Yunnan) of 0.5g, the rhizosphere soil sample of paddy rice (Foochow, Fujian) respectively, place the aseptic centrifuge tube of 2ml, add 100 μ l 1M Tris-HCl (pH=5.5), add V
1μ l aseptic double-distilled water (V
1=900 μ l-V
2), add V
2μ l 0.2M Al
2(SO
4)
3(V
2=60～300, because the content difference of impurity such as different regions or different planting material rhizosphere soil soil ulmin, according to embodiment 1 described each the suitableeest Al of step checking for examination soil
2(SO
4)
3Volume, so the Al that sugarcane soil adds in this example
2(SO
4)
3Volume be 150 μ l, the ground yellow soil adds 75 μ l, Root of Heterophylly Faalsestarwort soil adds 120ul, tobacco soil adds 120ul, paddy soil adds 150ul), vortex concussion 30s; Add 1/3 V
2Volume 4M NaOH adds V
3Volume 0.1M Tris-HCl (pH=8) (V
3=1300-4/3 V
2-V
1), vortex concussion 30s, regulate the soil solution pH value to 8 or more than, the centrifugal 2min of 3500 rpm removes supernatant and calculates supernatant volume V with liquid-transfering gun
4(1050 μ l).
(2) soil microorganisms lysis:
Add V
50.1M Tris-HCl (the pH=8) (V of (400 μ l) volume
5=V
4-650 μ l), add 0.5g 0.5mm granulated glass sphere, 0.3g 0.1mm granulated glass sphere and 1 4mm granulated glass sphere add 325 μ l 10%SDS (pH=8), 325 μ l EB (pH=8), vortex concussion 30s, ice bath 1min, vortex concussion 1min, ice bath 5min, vortex concussion 1min, 4 ℃ of 11000 centrifugal 1min of rpm.
Wherein EB (pH=8) solution preparation: take by weighing 2.416g LiCl, 1.211g Tris, 3.506g EDTA adds the distilled water dissolving of proper volume and regulates pH=8.0, is settled to 100ml.
(3) the free and precipitation of nucleic acid:
Get 750 μ l supernatants to the 1.5ml centrifuge tube, add 750 μ l phenol: chloroform: primary isoamyl alcohol (volume ratio=25:24:1), the vortex concussion, ice bath 5min, during per minute concussion 1 time, 4 ℃ of 16000 centrifugal 15min of rpm.Supernatant is transferred to 1.5ml centrifuge tube (0.1%DEPC soaks the back autoclaving), adds isopyknic chloroform: primary isoamyl alcohol (volume ratio=24:1), vortex concussion, 4 ℃ of 16000 centrifugal 15min of rpm.Supernatant is transferred to new 1.5ml centrifuge tube (0.1%DEPC soaks the back autoclaving), adds isopyknic chloroform: primary isoamyl alcohol (volume ratio=24:1), vortex concussion, 4 ℃ of 16000 centrifugal 15min of rpm.Supernatant is transferred to new 1.5ml centrifuge tube (0.1%DEPC soaks the back autoclaving), adds the Virahol of 0.7 times of volume, 0.1 times of volume 3M sodium-acetate (pH=5.5), 4 ℃ leave standstill 3h, 4 ℃ of 18000 centrifugal 60min of rpm.Remove supernatant, precipitation 5ml 75% ethanol (preparation of 0.1%DEPC water) washed twice, 4 ℃ of wash conditions, the centrifugal 5min of 18000 rpm.Remove supernatant, be deposited in and dry up in the aseptic technique platform and, get the aqueous solution that 5 μ l contain DNA and RNA, electrophoresis detection in 1% sepharose with 30 μ l 0.1%DEPC water dissolution.Electrophoresis result is seen Fig. 3, the DNA of UV spectrophotometer measuring different soils sample and the purity and the concentration of the RNA aqueous solution, and the result is as follows: the DNA of sugarcane soil extract and RNA aqueous solution purity are OD
260/ OD
280=1.71, concentration is 7.15 μ g/ μ l; The DNA of glutinous rehmannia soil extract and RNA aqueous solution purity are OD
260/ OD
280=1.72, concentration is 12.24 μ g/ μ l; The DNA of tobacco soil extract and RNA aqueous solution purity are OD
260/ OD
280=1.69, concentration is 9.95 μ g/ μ l; The DNA of Root of Heterophylly Faalsestarwort soil extract and RNA aqueous solution purity are OD
260/ OD
280=1.69, concentration is 8.98 μ g/ μ l; DNA and RNA aqueous solution purity that paddy soil extracts are OD
260/ OD
280=1.71, concentration is 11.35 μ g/ μ l.
Claims (5)
1. the extracting method of a soil microbe genome DNA and total RNA comprises the removal of pedotheque impurity, soil microorganisms lysis, the free precipitation that reaches of nucleic acid, and it is characterized in that: described method specifically may further comprise the steps:
(1) removal of pedotheque impurity: the fresh crop rhizosphere soils sample that takes by weighing 0.2～1.0g adds Tris-HCl, the V of 40～200 μ l 1M pH=5.5 in the aseptic centrifuge tube of 2ml
1μ l aseptic double-distilled water and V
2μ l 0.2M Al
2(SO
4)
3The aqueous solution, wherein V
1=900 μ l-V
2, V
2=60～300 μ l, vortex concussion 30s; Add 1/3 V then
2Volume 4M NaOH solution, and V
3The Tris-HCl of volume 0.1M pH=8, wherein V
3=1300-4/3 V
2-V
1, vortex concussion 30s, with pH value 〉=8 of the 4M NaOH solution adjusting soil solution, the centrifugal 2min of 3500rpm removes supernatant and calculates supernatant volume V with liquid-transfering gun
4
(2) soil microorganisms lysis: in the centrifugal precipitation that obtains of step (1), add V
5The Tris-HCl of the 0.1M pH=8 of volume, wherein V
5=V
4-650 μ l, add 0.5～0.8g 0.5mm granulated glass sphere again, 0.3～0.5g 0.1mm granulated glass sphere and 1～2 4mm granulated glass sphere, and then add the SDS aqueous solution of 325 μ l, 10% pH=8, the EB solution of 325 μ l pH=8, vortex concussion 30s, ice bath 1min, and then vortex concussion 1min, ice bath 5min, vortex concussion 1min once more, 4 ℃ of centrifugal 1min of 11000rpm then, it is standby to get supernatant;
(3) the free and precipitation of nucleic acid: get supernatant that 600～800 μ l steps (2) obtain to the aseptic centrifuge tube of 1.5ml, add and the isopyknic phenol of supernatant, chloroform and primary isoamyl alcohol mixed solution, phenol wherein: chloroform: primary isoamyl alcohol volume ratio=25:24:1, the vortex concussion, ice bath 5min, per minute concussion during this time 1 time, last 4 ℃ of centrifugal 15min of 16000rpm; Supernatant after centrifugal is transferred to the 1.5ml centrifuge tube, adds and the isopyknic chloroform of supernatant and primary isoamyl alcohol mixed solution, wherein chloroform: primary isoamyl alcohol volume ratio=24:1, the vortex concussion, 4 ℃ of centrifugal 15min of 16000rpm get supernatant; Supernatant is transferred to new 1.5ml centrifuge tube, adds and the isopyknic chloroform of supernatant and primary isoamyl alcohol mixed solution, wherein chloroform: primary isoamyl alcohol volume ratio=24:1, the vortex concussion, 4 ℃ of centrifugal 15min of 16000rpm get supernatant; Supernatant is transferred to another 1.5ml centrifuge tube, adds the Virahol of 0.7 times of supernatant volume and the sodium-acetate of 0.1 times of supernatant volume 3M pH=5.5,4 ℃ leave standstill 3h or-20 ℃ of precipitations 10min, 4 ℃ of centrifugal 60min of 18000rpm then; Remove supernatant, precipitation 5ml 75% ethanol washed twice under 4 ℃ of temperature, the centrifugal 5min of 18000rpm then; Remove supernatant, be deposited in and dry up in the aseptic technique platform and with 30 μ l 0.1%DEPC water dissolution, obtain containing DNA and the total solution of RNA ,-80 ℃ of preservations.
2. the extracting method of soil microbe genome DNA according to claim 1 and total RNA, it is characterized in that: described method specifically may further comprise the steps:
(1) removal of pedotheque impurity: the fresh crop rhizosphere soils sample that takes by weighing 0.5g adds Tris-HCl, the V of 100 μ l 1M pH=5.5 in the aseptic centrifuge tube of 2ml
1μ l aseptic double-distilled water and V
2μ l 0.2M Al
2(SO
4)
3The aqueous solution, wherein V
1=900 μ l-V
2, V
2=60～300 μ l, vortex concussion 30s; Add 1/3 V then
2Volume 4M NaOH solution, and V
3The Tris-HCl of volume 0.1M pH=8, wherein V
3=1300-4/3 V
2-V
1, vortex concussion 30s, with pH value 〉=8 of the 4M NaOH solution adjusting soil solution, the centrifugal 2min of 3500rpm removes supernatant and calculates supernatant volume V with liquid-transfering gun
4
(2) soil microorganisms lysis: in the centrifugal precipitation that obtains of step (1), add V
5The Tris-HCl of the 0.1M pH=8 of volume, wherein V
5=V
4-650 μ l, add 0.5g 0.5mm granulated glass sphere again, 0.3g 0.1mm granulated glass sphere and 1 4mm granulated glass sphere, and then add the SDS aqueous solution of 325 μ l, 10% pH=8, the EB solution of 325 μ l pH=8, vortex concussion 30s, ice bath 1min, and then vortex concussion 1min, ice bath 5min, vortex concussion 1min once more, 4 ℃ of centrifugal 1min of 11000rpm then, it is standby to get supernatant;
(3) the free and precipitation of nucleic acid: get supernatant that 750 μ l steps (2) obtain to the aseptic centrifuge tube of 1.5ml, add and the isopyknic phenol of supernatant, chloroform and primary isoamyl alcohol mixed solution, phenol wherein: chloroform: primary isoamyl alcohol volume ratio=25:24:1, the vortex concussion, ice bath 5min, per minute concussion during this time 1 time, last 4 ℃ of centrifugal 15min of 16000rpm; Supernatant after centrifugal is transferred to the 1.5ml centrifuge tube, adds and the isopyknic chloroform of supernatant and primary isoamyl alcohol mixed solution, wherein chloroform: primary isoamyl alcohol volume ratio=24:1, the vortex concussion, 4 ℃ of centrifugal 15min of 16000rpm get supernatant; Supernatant is transferred to new 1.5ml centrifuge tube, adds and the isopyknic chloroform of supernatant and primary isoamyl alcohol mixed solution, wherein chloroform: primary isoamyl alcohol volume ratio=24:1, the vortex concussion, 4 ℃ of centrifugal 15min of 16000rpm get supernatant; Supernatant is transferred to another 1.5ml centrifuge tube, adds the Virahol of 0.7 times of supernatant volume and the sodium-acetate of 0.1 times of supernatant volume 3M pH=5.5,4 ℃ leave standstill 3h or-20 ℃ of precipitations 10min, 4 ℃ of centrifugal 60min of 18000rpm then; Remove supernatant, precipitation 5ml 75% ethanol washed twice under 4 ℃ of temperature, the centrifugal 5min of 18000rpm then; Remove supernatant, be deposited in and dry up in the aseptic technique platform and with 30 μ l 0.1%DEPC water dissolution, obtain containing DNA and the total solution of RNA ,-80 ℃ of preservations.
3. the extracting method of soil microbe genome DNA according to claim 1 and 2 and total RNA is characterized in that: the described 1.5ml centrifuge tube of step (3) is earlier with 0.1%DEPC water logging bubble back autoclaving.
4. the extracting method of soil microbe genome DNA according to claim 1 and 2 and total RNA, it is characterized in that: being formulated as of step (2) described EB solution: take by weighing 2.416g LiCl, 1.211g Tris, 3.506g EDTA, add the distilled water dissolving and regulate pH=8.0, be settled to 100ml with 4M NaOH solution.
5. the extracting method of soil microbe genome DNA according to claim 1 and 2 and total RNA is characterized in that: described 75% ethanol of step (3) is prepared with 0.1%DEPC water.
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