CN103509787A - Method for extracting total genomic DNA from acidulated heavy metal tailings - Google Patents
Method for extracting total genomic DNA from acidulated heavy metal tailings Download PDFInfo
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Abstract
The invention discloses a method for extracting total genomic DNA from acidulated heavy metal tailings. The method comprises the following steps: adding a TE1 buffer solution to the acidulated heavy metal tailings; conducting vortex shaking; conducting a centrifuging process; adding a TE2 extracting and buffer solution, and lysozyme; conducting water-bath treatment; cooling the mixed solution I in liquid nitrogen and putting the mixed solution I in boiling water bath, repeating the operation for many times; adding SDS and protease K; after digestion, conducting the centrifuging process; taking the supernate after centrifuging; sequentially adding pre-cooled 8 mol/L Kac to the supernate; conducing ice-bath treatment; conducting the centrifuging process; adding phenol, chloroform and isoamyl alcohol; conducting the centrifuging process; then adding chloroform and isoamyl alcohol; conducting the centrifuging process; adding 3 mol/L NaAc and pre-cooled isopropyl alcohol; conducting the centrifuging process; discarding the supernate after conducting the centrifuging process; washing with an ethyl alcohol; precipitating; airing naturally; adding TE to dissolve the precipitate. The method is easy to operate, and can effectively avoid impacts from heavy metal, high salinity, low pH and the like of the acidulated heavy metal tailings. The total genomic DNA extracted according to the method is clear, high in integrity and purity, and low in impurity content.
Description
Technical field
The present invention relates to a kind for the treatment of process of acidifying heavy metal mine tailing, relate in particular to a kind of method of extracting genome DNA in acidifying heavy metal mine tailing.
Background technology
It is the basis of round pcr, restriction enzyme, the biological study of molecular hybridization equimolecular that genome DNA is extracted, acidifying heavy metal mine tailing has it with respect to soil environment more specifically, and main manifestations is high salinity, heavy metal kind is many and content is high, high-sulfate content and low pH.With this understanding, in acidifying heavy metal mine tailing, the genome DNA total amount soil of comparing will lack, and the activity of N,O-Diacetylmuramidase and other enzymes is suppressed.
In prior art, common soil genome DNA method steps is as follows:
(1) get 1.0g soil sample, pour in the 50mL centrifuge tube of sterilizing, [TENPP damping fluid forms: 20mmol/L EDTA(ethylenediamine tetraacetic acid (EDTA)) to add 10mLTENPP damping fluid, 50mmol/L Tris(Tutofusin tris), 1%PVPP(cross-linked polyvinylpyrrolidone), 100mmol/L NaCl(sodium-chlor), pH10.0] with shaker vibration several minutes, make soil sample suspend and fully mix.
(2) the centrifugal 5min of 10000r/min, abandons supernatant, and repeated washing 3-5 time is substantially colourless to supernatant liquor.
(3) add 5mL PBS damping fluid [damping fluid forms: 2.7mmol/L KCl(Repone K), 100mmol/L NaCl, 2mmol/L KH
2pO
4(potassium primary phosphate), 10mmol/L Na
2hPO
4(Sodium phosphate dibasic), pH7.4] rinsing, the centrifugal 5min of 10000r/min, abandons supernatant.
(4) add 13.5mL DNA extraction damping fluid [100mmol/L Tris, 100mmol/L EDTA, 100mmol/L Na
3pO
4(sodium phosphate), 1.5mol/L NaCl, 1%CTAB (cetyl trimethylammonium bromide), pH8.0], mix rear liquid nitrogen multigelation 3 times, add 100 μ L Proteinase Ks (25mg/mL) and 200 μ L N,O-Diacetylmuramidase (50mg/mL, pH8.0), 37 ℃ of water-bath 30min, during put upside down and mix 3 times.
(5) add 2mL20%SDS(Sodium dodecylbenzene sulfonate), 65 ℃ of water-bath 2h, during put upside down and mix 5-6 time, 8000r/min room temperature 15min, gets supernatant to new pipe.
(6) with equal-volume chloroform isoamyl alcohol (chloroform: primary isoamyl alcohol volume ratio is 24:1) extracting, sucking-off water layer, adds the Virahol of 0.6 times of volume, 4 ℃ of precipitation 1h or longer.
(7) 11000r/min4 ℃ of centrifugal 20min, precipitation DNA, removes supernatant, with 70% ethanol rinsing, 11000r/min4 ℃ of centrifugal 5min, and repeat rinsing 1 time, be placed in Bechtop and dry up.
(8) after dry, with 100 μ L ddH
2o(redistilled water) [containing RNase (rnase) 20mg/mL] dissolving, proceeds to 1.5mL centrifuge tube, places 30min for 37 ℃.
Get 3 μ L and detect for 0.8% agarose gel electrophoresis, result as shown in Figure 1, shows that above-mentioned soil DNA extracting method of the prior art can not extract the DNA in acidifying heavy metal mine tailing effectively.
Summary of the invention
The object of this invention is to provide a kind of method of extracting genome DNA in acidifying heavy metal mine tailing, the method is simple to operate, can effectively avoid the impact of the heavy metal of acidifying heavy metal mine tailing, high salinity, low pH etc., the DNA band that extracts is clear, integrity good, purity is high, foreign matter content is few, can be used for PCR detects, PCR specificity product is obvious, can meet the needs of molecular biology experiment.
The object of the invention is to be achieved through the following technical solutions:
The method of genome DNA in extraction acidifying heavy metal mine tailing of the present invention, comprises step:
A, in acidifying heavy metal mine tailing, add TE1 damping fluid, vortex vibration centrifugal treating 1 to 3 time, abandon supernatant liquor;
B, add TE2 Extraction buffer and N,O-Diacetylmuramidase, put into 37 ℃ of thermostat water bath water bath processing;
C, cooling in liquid nitrogen after, in boiling water bath, place to process, so repeatedly;
D, add SDS and Proteinase K, put into 65 ℃ of thermostat water baths and digest, carry out centrifugal treating after being down to normal temperature;
E, get the 8mol/L KAc(Potassium ethanoate that supernatant liquor after centrifugal in step D adds precooling) carry out, after ice bath, carrying out centrifugal treating;
F, get the supernatant liquor of step e after centrifugal and add phenol chloroform isoamyl alcohol, carry out centrifugal treating;
G, get the supernatant liquor of step F after centrifugal and add chloroform isoamyl alcohol, carry out centrifugal treating;
H, get the supernatant liquor of step G after centrifugal and add 3mol/L NaAc(sodium-acetate) and pre-cold isopropanol, precipitation at room temperature 2h;
I, carry out after centrifugal treating, discard the supernatant liquor after centrifugal, by washing with alcohol precipitation 3 times, naturally dry, add TE dissolution precipitation.
As seen from the above technical solution provided by the invention, in the extraction acidifying heavy metal mine tailing that the embodiment of the present invention provides, the method for genome DNA adds TE1 damping fluid in acidifying heavy metal mine tailing, and vortex vibration centrifugal treating 1 to 3 time, abandon supernatant liquor; Add TE2 Extraction buffer and N,O-Diacetylmuramidase, put into 37 ℃ of thermostat water bath water bath processing; After cooling in liquid nitrogen, in boiling water bath, place and process, so repeatedly; Add SDS and Proteinase K, put into 65 ℃ of thermostat water baths and digest, carry out centrifugal treating after being down to normal temperature; The supernatant liquor of getting after centrifugal in step D adds the 8mol/LKAc of precooling to carry out, after ice bath, carrying out centrifugal treating; Get the supernatant liquor of step e after centrifugal and add phenol chloroform isoamyl alcohol, carry out centrifugal treating; Get the supernatant liquor of step F after centrifugal and add chloroform isoamyl alcohol, carry out centrifugal treating; Get the supernatant liquor of step G after centrifugal and add 3mol/L NaAc and pre-cold isopropanol, precipitation at room temperature 2h; Carry out after centrifugal treating, discard the supernatant liquor after centrifugal, by washing with alcohol precipitation 3 times, naturally dry, add TE dissolution precipitation.Simple to operate, can effectively avoid the impact of the heavy metal of acidifying heavy metal mine tailing, high salinity, low pH etc., the DNA band of extraction is clear, integrity good, purity is high, foreign matter content is few, can be used for PCR and detects, PCR specificity product is obvious, can meet the needs of molecular biology experiment.
Accompanying drawing explanation
Fig. 1 is used normal soil extracting method to extract 0.8% agarose gel electrophoresis schematic diagram of acidifying heavy metal mine tailing genome DNA in prior art;
Fig. 2 is 0.8% agarose gel electrophoresis schematic diagram of acidifying heavy metal mine tailing genome DNA in the embodiment of the present invention one;
Fig. 3 is the 16S rRNA amplification PCR product electrophoresis schematic diagram of acidifying heavy metal mine tailing genome DNA in the embodiment of the present invention three.
Embodiment
To be described in further detail the embodiment of the present invention below.
The method of genome DNA in extraction acidifying heavy metal mine tailing of the present invention, its preferably embodiment comprise step:
A, in acidifying heavy metal mine tailing, add TE1 damping fluid, vortex vibration centrifugal treating 1 to 3 time, abandon supernatant liquor;
B, add TE2 Extraction buffer and N,O-Diacetylmuramidase, put into 37 ℃ of thermostat water bath water bath processing;
C, cooling in liquid nitrogen after, in boiling water bath, place to process, so repeatedly;
D, add SDS(Sodium dodecylbenzene sulfonate) and Proteinase K, put into 65 ℃ of thermostat water baths and digest, carry out centrifugal treating after being down to normal temperature;
E, the supernatant liquor of getting after centrifugal in step D add the 8mol/L KAc of precooling to carry out, after ice bath, carrying out centrifugal treating;
F, get the supernatant liquor of step e after centrifugal and add phenol chloroform isoamyl alcohol, carry out centrifugal treating;
G, get the supernatant liquor of step F after centrifugal and add chloroform isoamyl alcohol, carry out centrifugal treating;
H, get the supernatant liquor of step G after centrifugal and add 3mol/L NaAc and pre-cold isopropanol, precipitation at room temperature 2h;
I, carry out after centrifugal treating, discard the supernatant liquor after centrifugal, by washing with alcohol precipitation 3 times, naturally dry, adding TE(TE is Tris-EDTA damping fluid, and market is bought, moiety: 10mmol/L Tris-HCl, 1mmol/L EDTA, PH=8.0) dissolution precipitation.
Specifically comprise step:
A, get 0.5g acidifying heavy metal mine tailing, be placed in 1.5mL centrifuge tube, add 1mL TE1 damping fluid, vortex vibration 5min, the centrifugal 5min of 6000r/min, abandons supernatant liquor, then adds 1mlTE1 damping fluid, vortex vibration 5min, the centrifugal 5min of 6000r/min, abandons supernatant liquor;
B, add the N,O-Diacetylmuramidase that 600 μ L TE2 Extraction buffers and 10 μ L concentration are 100mg/mL, put into 37 ℃ of water-bath 1h of thermostat water bath, every 10min puts upside down and mixes once;
C, in liquid nitrogen cooling 5min, in boiling water bath, place rapidly 5min, so repeat 3 times;
D, add 120 μ L20%SDS and 10 μ L100mg/mL Proteinase Ks, put into 65 ℃ of digestion 1h of thermostat water bath, during every 10min put upside down and mix once, be down to after normal temperature the centrifugal 10min of 8000r/min;
E, get the 8mol/L KAc that supernatant liquor after centrifugal in step D adds the precooling of 0.2 times of volume, ice bath 20min, 10000r/min4 ℃ of centrifugal 10min;
F, get the supernatant liquor of step e after centrifugal and add isopyknic phenol chloroform isoamyl alcohol (phenol: chloroform: primary isoamyl alcohol volume ratio is 25:24:1), the centrifugal 10min of 12000r/min;
G, get the supernatant liquor of step F after centrifugal and add isopyknic chloroform isoamyl alcohol (chloroform: primary isoamyl alcohol volume ratio is 24:1), the centrifugal 10min of 12000r/min;
H, get the supernatant liquor of step G after centrifugal and add the 3mol/L NaAc of 0.1 times of volume and the pre-cold isopropanol of 0.6 times of volume, precipitation at room temperature 2h;
I, 14000r/min4 ℃ of centrifugal 10min, discards the supernatant liquor after centrifugal, and the washing with alcohol that is 70% by 500 μ L concentration precipitation 3 times, dries naturally, adds 50 μ LTE dissolution precipitations.
Described TE1 buffer formulation: 0.1mol/L phosphoric acid buffer, 3mol/L EDTA, 0.1mol/L Tris-HCl, pH=8.0:
Described TE2 Extraction buffer formula: 0.1mol/L phosphoric acid buffer, 3mol/L EDTA, 0.1mol/L Tris-HCl, 1.5mol/L NaCl, 1%CTAB, pH=8.0;
The extraction of genome DNA in acidifying heavy metal mine tailing and the difference of the extraction of normal soil are, the heavy metal kind of acidifying heavy metal mine tailing is many, content is high, high salinity, is acid, and the genome DNA content in acidifying heavy metal mine tailing is lower than normal soil.Heavy metal in acidifying heavy metal mine tailing and sour environment can suppress the activity of N,O-Diacetylmuramidase, obtain the genome DNA of high density, first need removal heavy metal, regulate specific conductivity and pH, avoid its impact on lysozyme activity.EDTA is chelating heavy metal effectively, before extracting mine tailing heavy metal, use the damping fluid with high density EDTA can remove mine tailing in a large amount of heavy metals.Use damping fluid of the present invention can effectively stablize pH and the specific conductivity of sample.For guaranteeing the purity of genome DNA, Proteinase K, SDS and phenol chloroform isoamyl alcohol (phenol: chloroform: primary isoamyl alcohol volume ratio is 25 ﹕ 24 ﹕ 1) can be removed a large amount of protein simultaneously, and CTAB, KAc be Polysaccharide removing class material effectively also.The present invention is simple to operate, and the DNA band of acquisition is clear, and integrity is good, and purity is high, and foreign matter content is few.
Specific embodiment:
Embodiment mono-, extracts acidifying heavy metal mine tailing genome DNA, comprises step:
(1) get 0.5g left and right acidifying heavy metal mine tailing, be placed in 1.5mL centrifuge tube, add 1mL TE1 damping fluid, vortex vibration 5min, the centrifugal 5min of 6000r/min, abandons supernatant liquor, then adds 1mlTE1 damping fluid, vortex vibration 5min, the centrifugal 5min of 6000r/min, abandons supernatant.
(2) add 600 μ L TE2 Extraction buffers and 10 μ L N,O-Diacetylmuramidases (100mg/mL) to put into 37 ℃ of water-bath 1h of thermostat water bath, every 10min puts upside down and mixes once.
(3) cooling 5min in liquid nitrogen puts into rapidly 5min in boiling water bath, so repeats 3 times.
(4) add 120 μ L20%SDS and 10 μ L100mg/mL Proteinase Ks, put into 65 ℃ of thermostat water baths digestion 1h, during every 10min put upside down and mix once.Be down to normal temperature, the centrifugal 10min of 8000r/min.
(5) get the 8mol/L KAc that above-mentioned supernatant liquor after centrifugal adds the precooling of 0.2 times of volume, ice bath 20min, 10000r/min4 ℃ of centrifugal 10min.
(6) get above-mentioned supernatant liquor after centrifugal and add isopyknic phenol chloroform isoamyl alcohol (phenol: chloroform: primary isoamyl alcohol volume ratio is 25 ﹕ 24 ﹕ 1), the centrifugal 10min of 12000r/min.
(7) get above-mentioned supernatant liquor after centrifugal and add isopyknic chloroform isoamyl alcohol (chloroform: primary isoamyl alcohol volume ratio is 24:1), the centrifugal 10min of 12000r/min.
(8) get above-mentioned supernatant liquor after centrifugal and add the 3mol/L NaAc of 0.1 times of volume and the pre-cold isopropanol of 0.6 times of volume, precipitation at room temperature 2h.
(9) 14000r/min4 ℃ of centrifugal 10min, discards above-mentioned supernatant liquor after centrifugal, by 500 μ L70% washing with alcohol precipitation 3 times, naturally dries, and adds 50 μ LTE dissolution precipitations.
Get 3 μ L and detect for 0.8% agarose gel electrophoresis, result as shown in Figure 1, shows that the DNA brightness obtaining by the inventive method is high, abundanter, more clear, and the repeatability of sample room is better.
Compare with soil DNA extracting method traditional in prior art, the DNA that present method is extracted has better stability, and the DNA concentration extracting is higher.
Embodiment bis-, the concentration of genome DNA and purity in working sample:
Utilize concentration and the purity of UV spectrophotometer measuring DNA, the light absorption value of the DNA solution that survey is extracted respectively when 260nm, 280nm, the light absorption value of the inventive method extraction DNA is as shown in table 1, the light absorption value of the DNA that traditional soil DNA extracting method obtains is as shown in table 2, calculates concentration and the purity of DNA sample.
Purity and the concentration of the acidifying heavy metal mine tailing genome DNA that table 1 the inventive method is extracted
Purity and the concentration of the acidifying heavy metal mine tailing genome DNA that in table 2 prior art, traditional soil DNA extracting method extracts
Conclusion: compare with soil DNA extracting method traditional in prior art, the DNA purity that the inventive method is extracted is higher, and concentration is larger, and effect is very desirable.
Embodiment tri-, and the PCR product of the 16S rRNA of acidifying heavy metal mine tailing genome DNA detects:
Use bacterium universal primer 27F:5 '-AGA GTT TGA TCC TGG CTC AG-3 ' and 1492R:5 '-GGTTAC CTT GTT ACG ACT T-3 '; PCR reaction system cumulative volume 50 μ L, 25 μ L Mix(10 * Buffer, dNTP, Mg
2+, TaqDNA polysaccharase), DNA profiling 2 μ L, each 2 μ L of primer, sterilizing distilled water 19 μ L.PCR reaction system: 94 ℃ of sex change 60s, 50 ℃ of annealing 30s, 72 ℃ are extended 60s, 35 circulations.Get 5 μ L products electrophoresis detection in 0.8% sepharose.
Result: as shown in Figure 3, sample is all realized effective amplification, and amplified production band is clear, the about 1500bp of fragment left and right, specific fragment length is consistent, illustrate that genome DNA in the acidifying heavy metal mine tailing that present method extracts can meet follow-up PCR and test etc.
The foregoing is only preferably embodiment of the present invention; but protection scope of the present invention is not limited to this; anyly be familiar with those skilled in the art in the technical scope that the present invention discloses, the variation that can expect easily or replacement, within all should being encompassed in protection scope of the present invention.Therefore, protection scope of the present invention should be as the criterion with the protection domain of claims.
Claims (3)
1. a method of extracting genome DNA in acidifying heavy metal mine tailing, is characterized in that, comprises step:
A, in acidifying heavy metal mine tailing, add TE1 damping fluid, vortex vibration centrifugal treating 1 to 3 time, abandon supernatant liquor;
B, add TE2 Extraction buffer and N,O-Diacetylmuramidase, put into 37 ℃ of thermostat water bath water bath processing;
C, cooling in liquid nitrogen after, in boiling water bath, place to process, so repeatedly;
D, add SDS and Proteinase K, put into 65 ℃ of thermostat water baths and digest, carry out centrifugal treating after being down to normal temperature;
E, the supernatant liquor of getting after centrifugal in step D add the 8mol/L KAc of precooling to carry out, after ice bath, carrying out centrifugal treating;
F, get the supernatant liquor of step e after centrifugal and add phenol chloroform isoamyl alcohol, carry out centrifugal treating;
G, get the supernatant liquor of step F after centrifugal and add chloroform isoamyl alcohol, carry out centrifugal treating;
H, get the supernatant liquor of step G after centrifugal and add 3mol/L NaAc and pre-cold isopropanol, precipitation at room temperature 2h;
I, carry out after centrifugal treating, discard the supernatant liquor after centrifugal, by washing with alcohol precipitation 3 times, naturally dry, add TE dissolution precipitation.
2. the method for genome DNA in extraction acidifying heavy metal mine tailing according to claim 1, is characterized in that, specifically comprises step:
A, get 0.5g acidifying heavy metal mine tailing, be placed in 1.5mL centrifuge tube, add 1mL TE1 damping fluid, vortex vibration 5min, the centrifugal 5min of 6000r/min, abandons supernatant liquor, then adds 1mL TE1 damping fluid, vortex vibration 5min, the centrifugal 5min of 6000r/min, abandons supernatant liquor;
B, add the N,O-Diacetylmuramidase that 600 μ L TE2 Extraction buffers and 10 μ L concentration are 100mg/ml, put into 37 ℃ of water-bath 1h of thermostat water bath, every 10min puts upside down and mixes once;
C, in liquid nitrogen cooling 5min, in boiling water bath, place rapidly 5min, so repeat 3 times;
D, add 120 μ L20%SDS and 10 μ L100mg/mL Proteinase Ks, put into 65 ℃ of digestion 1h of thermostat water bath, during every 10min put upside down and mix once, be down to after normal temperature the centrifugal 10min of 8000r/min;
E, get the 8mol/L KAc that supernatant liquor after centrifugal in step D adds the precooling of 0.2 times of volume, ice bath 20min, 10000r/min4 ℃ of centrifugal 10min;
F, get the supernatant liquor of step e after centrifugal and add isopyknic phenol: chloroform: primary isoamyl alcohol volume ratio is the phenol chloroform isoamyl alcohol of 25 ﹕ 24 ﹕ 1, the centrifugal 10min of 12000r/min;
G, get the supernatant liquor of step F after centrifugal and add isopyknic chloroform: primary isoamyl alcohol volume ratio is the chloroform isoamyl alcohol of 24 ﹕ 1, the centrifugal 10min of 12000r/min;
H, get the supernatant liquor of step G after centrifugal and add the 3mol/L NaAc of 0.1 times of volume and the pre-cold isopropanol of 0.6 times of volume, precipitation at room temperature 2h;
I, 14000r/min4 ℃ of centrifugal 10min, discards the supernatant liquor after centrifugal, and the washing with alcohol that is 70% by 500 μ L concentration precipitation 3 times, dries naturally, adds 50 μ LTE dissolution precipitations.
3. the method for genome DNA in extraction acidifying heavy metal mine tailing according to claim 1 and 2, is characterized in that:
Described TE1 buffer formulation: 0.1mol/L phosphoric acid buffer, 3mol/L EDTA, 0.1mol/L Tris-HCl, pH=8.0:
Described TE2 Extraction buffer formula: 0.1mol/L phosphoric acid buffer, 3mol/L EDTA, 0.1mol/L Tris-HCl, 1.5mol/L NaCl, 1%CTAB, pH=8.0.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110295162A (en) * | 2019-06-17 | 2019-10-01 | 广东省生态环境技术研究所 | A kind of DNA extraction reagent and extracting method for Fe-mn Nodules of Soils microorganism |
| CN110295163A (en) * | 2019-06-17 | 2019-10-01 | 广东省生态环境技术研究所 | The extraction reagent and extracting method of The Rhizosphere of Rice iron membrane micro DNA |
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| 吕志堂等: "磁铁矿尾矿宏基因组DNA提取方法的初步研究", 《安徽农业科学》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110295162A (en) * | 2019-06-17 | 2019-10-01 | 广东省生态环境技术研究所 | A kind of DNA extraction reagent and extracting method for Fe-mn Nodules of Soils microorganism |
| CN110295163A (en) * | 2019-06-17 | 2019-10-01 | 广东省生态环境技术研究所 | The extraction reagent and extracting method of The Rhizosphere of Rice iron membrane micro DNA |
| CN110295163B (en) * | 2019-06-17 | 2022-02-11 | 广东省科学院生态环境与土壤研究所 | Reagent and method for extracting rice rhizosphere iron membrane microorganism DNA |
| CN110295162B (en) * | 2019-06-17 | 2022-03-29 | 广东省科学院生态环境与土壤研究所 | DNA extraction reagent and extraction method for soil iron-manganese nodule microorganisms |
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