CN104560955A - Method for extracting genomic DNA of soil microorganism by using CTAB(cetyl trimethyl ammonium bromide) - Google Patents
Method for extracting genomic DNA of soil microorganism by using CTAB(cetyl trimethyl ammonium bromide) Download PDFInfo
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Abstract
The invention discloses a method for extracting genomic DNA of a soil microorganism by using a CTAB(cetyl trimethyl ammonium bromide). The method comprises the following steps: removing soil impurities and crushing cells of the microorganism; abstracting impure protein; adsorbing the DNA by a DNA adsorption column and removing impurities; eluting the adsorbed DNA. Compared with a traditional method, unique deproteinization liquid and rinse liquid are provided by the method disclosed by the invention to remove the impurities, and the genomic DNA of the soil microorganism can be quickly extracted by the method, so that plenty of extraction time is saved, besides the experiment steps are simplified, and the genomic DNA of the soil microorganism can be more effectively, more quickly and more economically extracted.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of method utilizing CTAB to extract soil microbe genome DNA.
Background technology
Microorganism is all tiny organisms that are invisible or that do not see, individual small, structure is simple, usually amplifies about 1000 times with opticmicroscope and electron microscope and just can see, the kind of Soil Microorganism is more, has bacterium, fungi, actinomycetes, algae and protozoon etc.Quantity is also very large, just has several hundred million to hundreds of hundred million in l gram of soil.Major part soil microorganisms is useful to crop growth, and they all have great effect to the formation growth of soil, material cycle and Fertility etc.
First, soil microorganisms can form Soil structure.Soil is not the simple combination of simple soil particle and chemical fertilizer, as the active group composition of soil, soil microorganisms is in the life process of oneself, by the oxygen of Metabolic activity and the exchange of carbonic acid gas, and the organic acid etc. of secretion contributes to soil particle and forms large crumb structure, the final soil formed truly.The fauna composition of soil microorganisms, biomass and vital movement thereof have substantial connection to the formation of soil and growth.Secondly, the most significant effect of soil microorganisms decomposes organic matter exactly, the undesirable root of such as crop loses leaf and organic fertilizer etc. in being manured into soil, only has the effect through soil microorganisms, could rot to decompose, discharge nutritive element, for crop utilization, and form soil ulmin, improve structure and the tilth of soil.Cause soil microbe genome DNA just because of materials such as the soil ulmin contained in soil, heavy metal, polyose, Polyphenols and extract difficulty, soil ulmin is owing to having the character similar with DNA, therefore can in the conventional extracting of DNA with DNA co-precipitation, the existence of soil ulmin minute quantity just can be reacted by suppression PCR consumingly.
Therefore need to develop convenient, fast, the practical method being applicable to soil microbial DNA and extracting.
Summary of the invention
The object of the present invention is to provide a kind of method utilizing CTAB to extract soil microbe genome DNA, described method more effectively, fast, economically can extract soil microbe genome DNA.
For achieving the above object, the present invention adopts following technical scheme:
Utilize CTAB to extract a method for soil microbe genome DNA, comprise the removal of soil impurity and the fragmentation of microorganism cells, extracting of foreign protein, the removal of DNA adsorption column adsorption of DNA and impurity, DNA wash-out, described method specifically comprises the following steps:
1) removal of soil impurity and the fragmentation of microorganism cells: take 1-10g soil in 50ml centrifuge tube, add 10-20ml lysate, concussion cracking 1-10min, in 60-70 DEG C of incubation 1-2h, every 5-10min concussion 1-2 time between incubation period; After incubation, the centrifugal 20-30min of 10000-12000rpm/min, get a supernatant liquor in aseptic centrifuge tube, add the 0.125-0.2 doubly 1-10mol/L Potassium ethanoate of a supernatant volume and the mass concentration 40-60% PEG-8 00 (PEG-800) of a 0.4-1 times of supernatant volume, in-20 DEG C of standing 30-60min, then the centrifugal 20-60min of 10000-12000rpm/min, 15-25mlCTAB-NaCl-EDTA solubilize precipitation is added, then in 65-80 DEG C of incubation 30-60min after abandoning waste liquid;
2) the extracting of foreign protein: after incubation, 15-20ml extract A extracting is added in above-mentioned centrifuge tube, the centrifugal 10-30min of 10000-13000rpm/min, get secondary supernatant liquor in aseptic centrifuge tube, add 15-20ml extract B extracting, the centrifugal 10-30min of 10000-13000rpm/min, gets three supernatant liquors in the centrifuge tube of sterilizing;
3) removal of DNA adsorption column adsorption of DNA and impurity: add in above-mentioned centrifuge tube and three isopyknic precipitation agents of supernatant liquor, add in DNA adsorption column after mixing, then the centrifugal 1-5min of 10000-13000rpm/min, abandon waste liquid, add 200-600 μ l protein liquid removal, then the centrifugal 1-5min of 10000-13000rpm/min, abandon waste liquid, add 200-600 μ l rinsing liquid, then the centrifugal 1-5min of 10000-13000rpm/min, abandon waste liquid, add 200-600 μ l rinsing liquid, then the centrifugal 1-5min of 10000-13000rpm/min, abandon waste liquid, DNA adsorption column is left standstill and dries up,
4) DNA wash-out: add 100-200 μ l elutriant in DNA adsorption column, then the centrifugal 1-5min of 10000-13000rpm/min, gained liquid is soil microbe genome DNA; Follow-uply get 2-7 μ l aqueous dna, in 1% sepharose, carry out electrophoresis detection;
Wherein, described lysate is by final concentration 0.25-1mol/L sodium-chlor (Nacl), final concentration 0.1-1mol/L ethylenediamine tetraacetic acid (EDTA) (EDTA), mass volume ratio 4-8% sodium lauryl sulphate (SDS) and final concentration 0.3-1mol/L guanidinium isothiocyanate composition;
Described CTAB-NaCl-EDTA solution is by mass volume ratio 1-5% cetyl trimethylammonium bromide (CTAB), and final concentration 1-2mol/L sodium-chlor (Nacl) and final concentration 0.1-0.5mol/L ethylenediamine tetraacetic acid (EDTA) (EDTA) form;
Described extract A refers to that volume ratio is phenol: chloroform: the mixed solution of primary isoamyl alcohol=25:24:1;
Described extract B refers to that volume ratio is chloroform: the mixed solution of primary isoamyl alcohol=24:1;
Described precipitation agent refers to mass concentration 70-80% ethanol;
Described protein liquid removal is made up of final concentration 3-6mol/L Guanidinium hydrochloride and final concentration 20-50mmol/L Tris-HCl, and pH=6-7, adds ethanol before using, and makes ethanol mass concentration in protein liquid removal be 30-40%;
Described rinsing liquid is made up of final concentration 10-30mmol/L NaCl and final concentration 1-5mmol/L Tris-HCl, and pH=7-8, adds ethanol after having adjusted pH, makes the ethanol mass concentration in rinsing liquid be 80-90%;
Described elutriant refers to 10-30mmol/L Tris-HCl, pH=8-9.
Described DNA adsorption column is Silicon moulds adsorption column, purchased from sky, Beijing bounties company.
Final concentration of the present invention refers to the final concentration of solution in working fluid, such as described lysate is by final concentration 0.25-1mol/L sodium-chlor (Nacl), final concentration 0.1-1mol/L ethylenediamine tetraacetic acid (EDTA) (EDTA), mass volume ratio 4-8% sodium lauryl sulphate (SDS) and final concentration 0.3-1mol/L guanidinium isothiocyanate composition, refer to that the concentration of Nacl in lysate is 0.25-1mol/L, the concentration of EDTA in lysate is 0.1-1mol/L, the mass volume ratio 4-8% of SDS in lysate, the concentration of guanidinium isothiocyanate in lysate is 0.3-1mol/L.
Further, described lysate is made up of final concentration 0.25mol/L Nacl, final concentration 0.1mol/L EDTA, mass volume ratio 4%SDS and final concentration 0.35mol/L guanidinium isothiocyanate.
Further, described CTAB-NaCl-EDTA solution is by mass volume ratio 2%CTAB, and final concentration 1.4mol/L Nacl and final concentration 0.1mol/L EDTA forms.
Further, described protein liquid removal is made up of final concentration 5mol/L Guanidinium hydrochloride and final concentration 20mmol/LTris-HCl, and pH=6.6, adds ethanol before using, and makes ethanol mass concentration in protein liquid removal be 38%.
Further, described rinsing liquid is made up of final concentration 20mmol/L NaCl and final concentration 2mmol/L Tris-HCl, and pH=7.5, adds ethanol after having adjusted pH, makes the ethanol mass concentration in rinsing liquid be 85%.
Further, described elutriant refers to 10mmol/L Tris-HCl, pH=8.5.
Further, described step 3) add rinsing liquid, centrifugal, after abandoning waste liquid, the centrifugal 1-5min of 10000-13000rpm/min, abandons waste liquid again, is left standstill by DNA adsorption column and dries up.
The present invention adopts above technical scheme, CTAB is utilized to extract soil microbe genome DNA, it is a kind of soil microorganisms genome C TAB extraction method of improvement, compared with traditional method, the method provides exclusive protein liquid removal and rinsing liquid to remove impurity, can rapid extraction to soil microbe genome DNA, not only save a large amount of extraction time, simplify experimental procedure simultaneously, more effectively, fast, economically can extract soil microbe genome DNA.
Accompanying drawing explanation
Fig. 1 is the electrophoresis detection figure of the soil microbe genome DNA that the inventive method is extracted:
Swimming lane 1 is the test-results of the inventive method extraction paddy soil microbe genome DNA;
Swimming lane 2 is the test-results of the inventive method extraction peanut soil microbe genome DNA;
Swimming lane 3 is the test-results of the inventive method extraction sugarcane soil microbe genome DNA;
Swimming lane 4 is the DL4500marker of TAKARA company.
Embodiment
Utilize CTAB to extract a method for soil microbe genome DNA, it comprises the following steps:
1) removal of soil impurity and the fragmentation of microorganism cells: take 1-10g soil in 50ml centrifuge tube, add 10-20ml lysate, concussion cracking 1-10min, in 60-70 DEG C of incubation 1-2h, every 5-10min concussion 1-2 time between incubation period; After incubation, the centrifugal 20-30min of 10000-12000rpm/min, get a supernatant liquor in aseptic centrifuge tube, add the 0.125-0.2 doubly 1-10mol/L Potassium ethanoate of a supernatant volume and the mass concentration 40-60% PEG-8 00 (PEG-800) of a 0.4-1 times of supernatant volume, in-20 DEG C of standing 30-60min, then the centrifugal 20-60min of 10000-12000rpm/min, 15-25mlCTAB-NaCl-EDTA solubilize precipitation is added, then in 65-80 DEG C of incubation 30-60min after abandoning waste liquid;
2) the extracting of foreign protein: after incubation, 15-20ml extract A extracting is added in above-mentioned centrifuge tube, the centrifugal 10-30min of 10000-13000rpm/min, get secondary supernatant liquor in aseptic centrifuge tube, add 15-20ml extract B extracting, the centrifugal 10-30min of 10000-13000rpm/min, gets three supernatant liquors in the centrifuge tube of sterilizing;
3) removal of DNA adsorption column adsorption of DNA and impurity: add in above-mentioned centrifuge tube and three isopyknic precipitation agents of supernatant liquor, add in DNA adsorption column after mixing, then the centrifugal 1-5min of 10000-13000rpm/min, abandon waste liquid, add 200-600 μ l protein liquid removal, then the centrifugal 1-5min of 10000-13000rpm/min, abandon waste liquid, add 200-600 μ l rinsing liquid, then the centrifugal 1-5min of 10000-13000rpm/min, abandon waste liquid, add 200-600 μ l rinsing liquid, then the centrifugal 1-5min of 10000-13000rpm/min, abandon waste liquid, then the centrifugal 1-5min of 10000-13000rpm/min, abandon waste liquid, DNA adsorption column is left standstill and dries up,
4) DNA wash-out: add 100-200 μ l elutriant in DNA adsorption column, then the centrifugal 1-5min of 10000-13000rpm/min, gained liquid is soil microbe genome DNA; Follow-uply get 2-7 μ l aqueous dna, in 1% sepharose, carry out electrophoresis detection;
Wherein, described lysate is by final concentration 0.25-1mol/L sodium-chlor (Nacl), final concentration 0.1-1mol/L ethylenediamine tetraacetic acid (EDTA) (EDTA), mass volume ratio 4-8% sodium lauryl sulphate (SDS) and final concentration 0.3-1mol/L guanidinium isothiocyanate composition;
Described CTAB-NaCl-EDTA solution is by mass volume ratio 1-5% cetyl trimethylammonium bromide (CTAB), and final concentration 1-2mol/L sodium-chlor (Nacl) and final concentration 0.1-0.5mol/L ethylenediamine tetraacetic acid (EDTA) (EDTA) form;
Described extract A refers to that volume ratio is phenol: chloroform: the mixed solution of primary isoamyl alcohol=25:24:1;
Described extract B refers to that volume ratio is chloroform: the mixed solution of primary isoamyl alcohol=24:1;
Described precipitation agent refers to mass concentration 70-80% ethanol;
Described protein liquid removal is made up of final concentration 3-6mol/L Guanidinium hydrochloride and final concentration 20-50mmol/L Tris-HCl, and pH=6-7, adds ethanol before using, and makes ethanol mass concentration in protein liquid removal be 30-40%;
Described rinsing liquid is made up of final concentration 10-30mmol/L NaCl and final concentration 1-5mmol/L Tris-HCl, and pH=7-8, adds ethanol after having adjusted pH, makes the ethanol mass concentration in rinsing liquid be 80-90%;
Described elutriant refers to 10-30mmol/L Tris-HCl, pH=8-9.
Described DNA adsorption column is Silicon moulds adsorption column, purchased from sky, Beijing bounties company.
Embodiment 1
Utilize CTAB to extract a method for soil microbe genome DNA, it comprises the following steps:
1) removal of soil impurity and the fragmentation of microorganism cells: take 5g paddy soil, peanut soil, sugarcane soil respectively in 50ml centrifuge tube, respectively add 20ml lysate, concussion cracking 5min, in 65 DEG C of incubation 1h, between incubation period, every 5min shakes 1 time; After incubation, the centrifugal 20min of 10000rpm/min, get a supernatant liquor in aseptic centrifuge tube, add the 5mol/L Potassium ethanoate of 0.125 times of supernatant volume and mass concentration 40% PEG-8 00 (PEG-800) of 0.42 times of supernatant volume, in-20 DEG C of standing 30min, then the centrifugal 20min of 10000rpm/min, adds 15ml CTAB-NaCl-EDTA solubilize precipitation, then in 65 DEG C of incubation 30min after abandoning waste liquid;
2) the extracting of foreign protein: after incubation, 15ml extract A extracting is added in above-mentioned centrifuge tube, the centrifugal 10min of 12000rpm/min, get secondary supernatant liquor in aseptic centrifuge tube, add 15ml extract B extracting, the centrifugal 10min of 12000rpm/min, gets three supernatant liquors in the centrifuge tube of sterilizing;
3) removal of DNA adsorption column adsorption of DNA and impurity: add in above-mentioned centrifuge tube and three isopyknic precipitation agents of supernatant liquor, add in DNA adsorption column after mixing, then the centrifugal 1min of 12000rpm/min, abandon waste liquid, add 500 μ l protein liquid removals, then the centrifugal 1min of 12000rpm/min, abandon waste liquid, add 500 μ l rinsing liquids, then the centrifugal 1-5min of 10000-13000rpm/min, abandon waste liquid, add 200-600 μ l rinsing liquid, then the centrifugal 1min of 12000rpm/min, abandon waste liquid, then the centrifugal 2min of 12000rpm/min, abandon waste liquid, DNA adsorption column is left standstill and dries up,
4) DNA wash-out: add 100 μ l elutriants in DNA adsorption column, then the centrifugal 2min of 12000rpm/min, gained liquid is soil microbe genome DNA; Follow-uply get 5 μ l aqueous dnas, in 1% sepharose, carry out electrophoresis detection;
Wherein, described lysate is by final concentration 0.25mol/L sodium-chlor (Nacl), final concentration 0.1mol/L ethylenediamine tetraacetic acid (EDTA) (EDTA), mass volume ratio 4% sodium lauryl sulphate (SDS) and final concentration 0.35mol/L guanidinium isothiocyanate composition;
Described CTAB-NaCl-EDTA solution is by mass volume ratio 2% cetyl trimethylammonium bromide (CTAB), and final concentration 1.4mol/L sodium-chlor (Nacl) and final concentration 0.1mol/L ethylenediamine tetraacetic acid (EDTA) (EDTA) form;
Described extract A refers to that volume ratio is phenol: chloroform: the mixed solution of primary isoamyl alcohol=25:24:1;
Described extract B refers to that volume ratio is chloroform: the mixed solution of primary isoamyl alcohol=24:1;
Described precipitation agent refers to mass concentration 70% ethanol;
Described protein liquid removal is made up of final concentration 5mol/L Guanidinium hydrochloride and final concentration 20mmol/L Tris-HCl, and pH=6.6, adds ethanol before using, and makes ethanol mass concentration in protein liquid removal be 38%;
Described rinsing liquid is made up of final concentration 20mmol/L NaCl and final concentration 2mmol/L Tris-HCl, and pH=7.5, adds ethanol after having adjusted pH, makes the ethanol mass concentration in rinsing liquid be 85%;
Described elutriant refers to 10mmol/L Tris-HCl, pH=8.5.
Embodiment 2
Utilize CTAB to extract a method for soil microbe genome DNA, it comprises the following steps:
1) removal of soil impurity and the fragmentation of microorganism cells: take 1g soil in 50ml centrifuge tube, add 10ml lysate, concussion cracking 1min, in 60 DEG C of incubation 2h, between incubation period, every 10min shakes 2 times; After incubation, the centrifugal 30min of 12000rpm/min, get a supernatant liquor in aseptic centrifuge tube, add the 1mol/L Potassium ethanoate of 0.2 times of supernatant volume and mass concentration 60% PEG-8 00 (PEG-800) of 0.4 times of supernatant volume, in-20 DEG C of standing 60min, then the centrifugal 60min of 12000rpm/min, adds 25ml CTAB-NaCl-EDTA solubilize precipitation, then in 70 DEG C of incubation 60min after abandoning waste liquid;
2) the extracting of foreign protein: after incubation, 20ml extract A extracting is added in above-mentioned centrifuge tube, the centrifugal 30min of 10000rpm/min, get secondary supernatant liquor in aseptic centrifuge tube, add 20ml extract B extracting, the centrifugal 30min of 10000rpm/min, gets three supernatant liquors in the centrifuge tube of sterilizing;
3) removal of DNA adsorption column adsorption of DNA and impurity: add in above-mentioned centrifuge tube and three isopyknic precipitation agents of supernatant liquor, add in DNA adsorption column after mixing, then the centrifugal 5min of 10000rpm/min, abandon waste liquid, add 200 μ l protein liquid removals, then the centrifugal 5min of 10000rpm/min, abandon waste liquid, add 200 μ l rinsing liquids, then centrifugal-the 5min of 10000rpm/min, abandon waste liquid, add 200 μ l rinsing liquids, then the centrifugal 5min of 10000rpm/min, abandon waste liquid, then the centrifugal 5min of 10000rpm/min, abandon waste liquid, DNA adsorption column is left standstill and dries up,
4) DNA wash-out: add 200 μ l elutriants in DNA adsorption column, then the centrifugal 5min of 10000rpm/min, gained liquid is soil microbe genome DNA; Follow-uply get 2-7 μ l aqueous dna, in 1% sepharose, carry out electrophoresis detection;
Wherein, described lysate is by final concentration 0.5mol/L sodium-chlor (Nacl), final concentration 0.5mol/L ethylenediamine tetraacetic acid (EDTA) (EDTA), mass volume ratio 6% sodium lauryl sulphate (SDS) and final concentration 0.3mol/L guanidinium isothiocyanate composition;
Described CTAB-NaCl-EDTA solution is by mass volume ratio 1% cetyl trimethylammonium bromide (CTAB), and final concentration 1mol/L sodium-chlor (Nacl) and final concentration 0.25mol/L ethylenediamine tetraacetic acid (EDTA) (EDTA) form;
Described extract A refers to that volume ratio is phenol: chloroform: the mixed solution of primary isoamyl alcohol=25:24:1;
Described extract B refers to that volume ratio is chloroform: the mixed solution of primary isoamyl alcohol=24:1;
Described precipitation agent refers to mass concentration 75% ethanol;
Described protein liquid removal is made up of final concentration 3mol/L Guanidinium hydrochloride and final concentration 35mmol/L Tris-HCl, and pH=7, adds ethanol before using, and makes ethanol mass concentration in protein liquid removal be 30%;
Described rinsing liquid is made up of final concentration 10mmol/L NaCl and final concentration 1mmol/L Tris-HCl, and pH=8, adds ethanol after having adjusted pH, makes the ethanol mass concentration in rinsing liquid be 80%;
Described elutriant refers to 20mmol/L Tris-HCl, pH=9.
Embodiment 3
Utilize CTAB to extract a method for soil microbe genome DNA, it comprises the following steps:
1) removal of soil impurity and the fragmentation of microorganism cells: take 10g soil in 50ml centrifuge tube, add 20ml lysate, concussion cracking 10min, in 70 DEG C of incubation 2h, between incubation period, every 5min shakes 1 time; After incubation, the centrifugal 25min of 10000rpm/min, get a supernatant liquor in aseptic centrifuge tube, add the 10mol/L Potassium ethanoate of 0.15 times of supernatant volume and mass concentration 40% PEG-8 00 (PEG-800) of 1 times of supernatant volume, in-20 DEG C of standing 40min, then the centrifugal 40min of 10000rpm/min, adds 20ml CTAB-NaCl-EDTA solubilize precipitation, then in 80 DEG C of incubation 45min after abandoning waste liquid;
2) the extracting of foreign protein: after incubation, 15ml extract A extracting is added in above-mentioned centrifuge tube, the centrifugal 20min of 13000rpm/min, get secondary supernatant liquor in aseptic centrifuge tube, add 15ml extract B extracting, the centrifugal 20min of 13000rpm/min, gets three supernatant liquors in the centrifuge tube of sterilizing;
3) removal of DNA adsorption column adsorption of DNA and impurity: add in above-mentioned centrifuge tube and three isopyknic precipitation agents of supernatant liquor, add in DNA adsorption column after mixing, then the centrifugal 2min of 13000rpm/min, abandon waste liquid, add 600 μ l protein liquid removals, then the centrifugal 2min of 13000rpm/min, abandon waste liquid, add 600 μ l rinsing liquids, then the centrifugal 2min of 13000rpm/min, abandon waste liquid, add 600 μ l rinsing liquids, then the centrifugal 2min of 13000rpm/min, abandon waste liquid, then the centrifugal 1min of 13000rpm/min, abandon waste liquid, DNA adsorption column is left standstill and dries up,
4) DNA wash-out: add 200 μ l elutriants in DNA adsorption column, then the centrifugal 1min of 13000rpm/min, gained liquid is soil microbe genome DNA; Follow-uply get 2-7 μ l aqueous dna, in 1% sepharose, carry out electrophoresis detection;
Wherein, described lysate is by final concentration 1mol/L sodium-chlor (Nacl), final concentration 1mol/L ethylenediamine tetraacetic acid (EDTA) (EDTA), mass volume ratio 8% sodium lauryl sulphate (SDS) and final concentration 1mol/L guanidinium isothiocyanate composition;
Described CTAB-NaCl-EDTA solution is by mass volume ratio 5% cetyl trimethylammonium bromide (CTAB), and final concentration 2mol/L sodium-chlor (Nacl) and final concentration 0.5mol/L ethylenediamine tetraacetic acid (EDTA) (EDTA) form;
Described extract A refers to that volume ratio is phenol: chloroform: the mixed solution of primary isoamyl alcohol=25:24:1;
Described extract B refers to that volume ratio is chloroform: the mixed solution of primary isoamyl alcohol=24:1;
Described precipitation agent refers to mass concentration 80% ethanol;
Described protein liquid removal is made up of final concentration 6mol/L Guanidinium hydrochloride and final concentration 50mmol/L Tris-HCl, and pH=6, adds ethanol before using, and makes ethanol mass concentration in protein liquid removal be 40%;
Described rinsing liquid is made up of final concentration 30mmol/L NaCl and final concentration 5mmol/L Tris-HCl, and pH=7, adds ethanol after having adjusted pH, makes the ethanol mass concentration in rinsing liquid be 90%;
Described elutriant refers to 30mmol/L Tris-HCl, pH=8.
The electrophoresis detection of soil microbe genome DNA in 1% sepharose extracted according to the method for embodiment 1 the results are shown in Figure 1, wherein swimming lane 1,2,3, be paddy soil, peanut soil, sugarcane soil microbe genome DNA test-results respectively, swimming lane 4 is the DL4500marker of TAKARA company.As can be seen from Figure 1, the present invention can extract soil microbe genome DNA.
The soil microbe genome DNA extracted, its purity OD
260nm/ OD
280nmratio is weighed, and purity is in table 1.
The purity of table 1 soil microbe genome DNA
As can be seen from Table 1, OD (260:280) scope of DNA is at 1.78-1.80.Therefrom can find out, present method can extract the higher soil microorganisms genome of purity.
Claims (7)
1. the method utilizing CTAB to extract soil microbe genome DNA, comprise the removal of soil impurity and the fragmentation of microorganism cells, extracting of foreign protein, the removal of DNA adsorption column adsorption of DNA and impurity, DNA wash-out, is characterized in that: described method specifically comprises the following steps:
1) removal of soil impurity and the fragmentation of microorganism cells
:take soil in centrifuge tube, in every 1-10g soil, add 10-20ml lysate, concussion cracking 1-10min, in 60-70 ° of C incubation 1-2h, every 5-10min concussion 1-2 time between incubation period; After incubation; the centrifugal 20-30min of 10000-12000rpm/min; get a supernatant liquor in aseptic centrifuge tube; add the 0.125-0.2 doubly 1-10mol/L Potassium ethanoate of a supernatant volume and the mass concentration 40-60% PEG-8 00 of a 0.4-1 times of supernatant volume; 30-60min is left standstill in-20 ° of C; then the centrifugal 20-60min of 10000-12000rpm/min, adds 15-25ml CTAB-NaCl-EDTA solution after abandoning waste liquid, then in 65-80 ° of C incubation 30-60min;
2) the extracting of foreign protein: add 15-20ml extract A extracting in above-mentioned centrifuge tube, the centrifugal 10-30min of 10000-13000rpm/min, get secondary supernatant liquor in aseptic centrifuge tube, add 15-20ml extract B extracting, the centrifugal 10-30min of 10000-13000rpm/min, gets three supernatant liquors in the centrifuge tube of sterilizing;
3) removal of DNA adsorption column adsorption of DNA and impurity: add in above-mentioned centrifuge tube and three isopyknic precipitation agents of supernatant liquor, add in DNA adsorption column after mixing, then the centrifugal 1-5min of 10000-13000rpm/min, abandon waste liquid, add 200-600 μ l protein liquid removal, then the centrifugal 1-5min of 10000-13000rpm/min, abandon waste liquid, add 200-600 μ l rinsing liquid, then the centrifugal 1-5min of 10000-13000rpm/min, abandon waste liquid, add 200-600 μ l rinsing liquid, then the centrifugal 1-5min of 10000-13000rpm/min, abandon waste liquid, DNA adsorption column is left standstill and dries up,
4) DNA wash-out: add 100-200 μ l elutriant in DNA adsorption column, then the centrifugal 1-5min of 10000-13000rpm/min, gained liquid is soil microbe genome DNA;
Described lysate by final concentration 0.25-1mol/L sodium-chlor, final concentration 0.1-1 mol/L ethylenediamine tetraacetic acid (EDTA), mass volume ratio 4-8% sodium lauryl sulphate and final concentration 0.3-1 mol/L guanidinium isothiocyanate composition;
Described CTAB-NaCl-EDTA solution is by mass volume ratio 1-5% cetyl trimethylammonium bromide, and final concentration 1-2 mol/L sodium-chlor and final concentration 0.1-0.5 mol/L ethylenediamine tetraacetic acid (EDTA) form;
Described extract A refers to that volume ratio is phenol: chloroform: the mixed solution of primary isoamyl alcohol=25:24:1;
Described extract B refers to that volume ratio is chloroform: the mixed solution of primary isoamyl alcohol=24:1;
Described precipitation agent refers to mass concentration 70-80% ethanol;
Described protein liquid removal is made up of final concentration 3-6mol/L Guanidinium hydrochloride and final concentration 20-50 mmol/L Tris-HCl, and pH=6-7, adds ethanol before using, and makes ethanol mass concentration in protein liquid removal be 30-40%;
Described rinsing liquid is made up of final concentration 10-30mmol/L NaCl and final concentration 1-5mmol/L Tris-HCl, and pH=7-8, adds ethanol after having adjusted pH, makes the ethanol mass concentration in rinsing liquid be 80-90%;
Described elutriant refers to 10-30mmol/L Tris-HCl, pH=8-9.
2. a kind of method utilizing CTAB to extract soil microbe genome DNA according to claim 1, it is characterized in that: described lysate is by final concentration 0.25mol/L sodium-chlor, final concentration 0.1 mol/L ethylenediamine tetraacetic acid (EDTA), mass volume ratio 4% sodium lauryl sulphate and final concentration 0.35 mol/L guanidinium isothiocyanate composition.
3. a kind of method utilizing CTAB to extract soil microbe genome DNA according to claim 1, it is characterized in that: described CTAB-NaCl-EDTA solution is by mass volume ratio 2% cetyl trimethylammonium bromide, and final concentration 1.4 mol/L sodium-chlor and final concentration 0.1mol/L ethylenediamine tetraacetic acid (EDTA) form.
4. a kind of method utilizing CTAB to extract soil microbe genome DNA according to claim 1, it is characterized in that: described protein liquid removal is made up of final concentration 5mol/L Guanidinium hydrochloride and final concentration 20 mmol/L Tris-HCl, pH=6.6, add ethanol before using, make ethanol mass concentration in protein liquid removal be 38%.
5. a kind of method utilizing CTAB to extract soil microbe genome DNA according to claim 1, it is characterized in that: described rinsing liquid is made up of final concentration 20mmol/L NaCl and final concentration 2mmol/L Tris-HCl, pH=7.5, add ethanol after having adjusted pH, make the ethanol mass concentration in rinsing liquid be 85%.
6. a kind of method utilizing CTAB to extract soil microbe genome DNA according to claim 1, is characterized in that: described elutriant refers to 10mmol/L Tris-HCl, pH=8.5.
7. a kind of method utilizing CTAB to extract soil microbe genome DNA according to claim 1, is characterized in that: described step 3) adds rinsing liquid, centrifugal, after abandoning waste liquid, the centrifugal 1-5min of 10000-13000rpm/min, abandons waste liquid again, is left standstill by DNA adsorption column and dries up.
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| CN104975004A (en) * | 2015-07-28 | 2015-10-14 | 福建师范大学 | Kit for rapidly extracting genomes of animal excrement by virtue of CTAB method |
| CN104975006A (en) * | 2015-07-28 | 2015-10-14 | 福建师范大学 | Kit for extracting fungal genome DNA |
| CN105039310A (en) * | 2015-07-28 | 2015-11-11 | 福建师范大学 | Kit for extracting agrobacterium tumefaciens plasmid DNA |
| CN105087547A (en) * | 2015-09-07 | 2015-11-25 | 昆明理工大学 | Method for extracting bacterial genomes in ores |
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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 |
| CN103789300A (en) * | 2014-02-26 | 2014-05-14 | 济南大学 | Extraction method of metagenome DNA (deoxyribonucleic acid) in activated sludge for treating epoxypropane saponification wastewater |
| CN104178481A (en) * | 2014-09-05 | 2014-12-03 | 福建师范大学 | Kit for extracting soil microbial genome DNA within 30 minute |
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| CN103789300A (en) * | 2014-02-26 | 2014-05-14 | 济南大学 | Extraction method of metagenome DNA (deoxyribonucleic acid) in activated sludge for treating epoxypropane saponification wastewater |
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| CN104975004A (en) * | 2015-07-28 | 2015-10-14 | 福建师范大学 | Kit for rapidly extracting genomes of animal excrement by virtue of CTAB method |
| CN104975006A (en) * | 2015-07-28 | 2015-10-14 | 福建师范大学 | Kit for extracting fungal genome DNA |
| CN105039310A (en) * | 2015-07-28 | 2015-11-11 | 福建师范大学 | Kit for extracting agrobacterium tumefaciens plasmid DNA |
| CN105087547A (en) * | 2015-09-07 | 2015-11-25 | 昆明理工大学 | Method for extracting bacterial genomes in ores |
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