CN103509787B - 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 of processing method of acidifying heavy metal mine tailing, particularly relate to a kind of method extracting genome DNA in acidifying heavy metal mine tailing.
Background technology
Genome DNA extraction is the basis of round pcr, restriction enzyme, the biological study of molecular hyridization equimolecular, acidifying heavy metal mine tailing has it relative to soil more specifically environment, 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, genome DNA total amount soil of comparing will lack, and the activity of lysozyme and other enzymes is suppressed.
In prior art, common soil genome DNA method step is as follows:
(1) 1.0g soil sample is got, pour in the 50mL centrifuge tube of sterilizing, add 10mLTENPP buffer solution [TENPP buffer solution form: 20mmol/L EDTA(ethylenediamine tetra-acetic acid), 50mmol/L Tris(trishydroxymethylaminomethane), 1%PVPP(PVPP), 100mmol/L NaCl(sodium chloride), pH10.0] vibrate several minutes with shaker, soil sample is suspended and fully mixing.
(2) the centrifugal 5min of 10000r/min, abandons supernatant, repeated washing 3-5 time, substantially colourless to supernatant.
(3) add 5mL PBS buffer solution [buffer solution form: 2.7mmol/L KCl(potassium chloride), 100mmol/LNaCl, 2mmol/L KH
2pO
4(potassium dihydrogen phosphate), 10mmol/L Na
2hPO
4(sodium hydrogen phosphate), pH7.4] rinsing, the centrifugal 5min of 10000r/min, abandons supernatant.
(4) 13.5mL DNA Extraction buffer [100mmol/L Tris, 100mmol/L EDTA, 100mmol/LNa is added
3pO
4(sodium phosphate), 1.5mol/L NaCl, 1%CTAB (softex kw), pH8.0], after mixing, liquid nitrogen multigelation 3 times, adds 100 μ L Proteinase Ks (25mg/mL) and 200 μ L lysozyme (50mg/mL, pH8.0), 37 DEG C of water-bath 30min, period puts upside down mixing 3 times.
(5) 2mL20%SDS(neopelex is added), 65 DEG C of water-bath 2h, period puts upside down mixing 5-6 time, 8000r/min room temperature 15min, gets supernatant to new pipe.
(6) with equal-volume chloroform isoamyl alcohol (chloroform: isoamyl alcohol volume ratio is 24:1) extracting, sucking-off water layer, adds the isopropyl alcohol of 0.6 times of volume, 4 DEG C of precipitation 1h or longer.
(7) 11000r/min4 DEG C of centrifugal 20min, precipitation DNA, removes supernatant, uses 70% ethanol rinse, 11000r/min4 DEG C of centrifugal 5min, and repeat rinsing 1 time, be placed in superclean bench and dry up.
(8) after drying, with 100 μ L ddH
2o(redistilled water) [containing RNase (ribalgilase) 20mg/mL] dissolving, proceed to 1.5mL centrifuge tube, place 30min for 37 DEG C.
Get 3 μ L to 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 extracting genome DNA in acidifying heavy metal mine tailing, the method is simple to operate, effectively can avoid the impact of the heavy metal of acidifying heavy metal mine tailing, high salinity, low pH etc., the DNA band extracted is clear, integrality good, purity is high, impurity content is few, can be used for PCR to detect, PCR specific 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 buffer solution, vortex oscillation centrifugal treating 1 to 3 time, abandon supernatant;
B, add TE2 Extraction buffer and lysozyme, put into 37 DEG C of thermostat water bath water bath processing;
C, in liquid nitrogen cooling after, in boiling water bath place process, so repeatedly;
D, add SDS and Proteinase K, put into 65 DEG C of thermostat water baths and digest, after being down to normal temperature, carry out centrifugal treating;
E, get centrifugal in step D after supernatant add the 8mol/L KAc(potassium acetate of precooling) carry out ice bath after, carry out centrifugal treating;
F, get step e centrifugal after supernatant add phenol chloroform isoamyl alcohol, carry out centrifugal treating;
G, get step F centrifugal after supernatant add chloroform isoamyl alcohol, carry out centrifugal treating;
H, get step G centrifugal after supernatant add 3mol/L NaAc(sodium acetate) and pre-cold isopropanol, precipitation at room temperature 2h;
I, carry out centrifugal treating after, discard centrifugal after supernatant, with ethanol washing 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 buffer solution in acidifying heavy metal mine tailing, vortex oscillation centrifugal treating 1 to 3 time, abandons supernatant; Add TE2 Extraction buffer and lysozyme, put into 37 DEG C of thermostat water bath water bath processing; In liquid nitrogen after cooling, in boiling water bath, place process, so repeatedly; Add SDS and Proteinase K, put into 65 DEG C of thermostat water baths and digest, after being down to normal temperature, carry out centrifugal treating; Get centrifugal in step D after the supernatant 8mol/LKAc that adds precooling carry out ice bath after, carry out centrifugal treating; Get step e centrifugal after supernatant add phenol chloroform isoamyl alcohol, carry out centrifugal treating; Get step F centrifugal after supernatant add chloroform isoamyl alcohol, carry out centrifugal treating; Get step G centrifugal after supernatant add 3mol/L NaAc and pre-cold isopropanol, precipitation at room temperature 2h; After carrying out centrifugal treating, discard centrifugal after supernatant, with ethanol washing precipitation 3 times, naturally dry, add TE dissolution precipitation.Simple to operate, effectively can 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, integrality good, purity is high, impurity content is few, can be used for PCR and detects, PCR specific product is obvious, can meet the needs of molecular biology experiment.
Accompanying drawing explanation
Fig. 1 is the 0.8% agarose gel electrophoresis schematic diagram using normal soil extracting method to extract 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 primer electrophoresis schematic diagram of acidifying heavy metal mine tailing genome DNA in the embodiment of the present invention three.
Detailed description of the invention
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 detailed description of the invention comprise step:
A, in acidifying heavy metal mine tailing, add TE1 buffer solution, vortex oscillation centrifugal treating 1 to 3 time, abandon supernatant;
B, add TE2 Extraction buffer and lysozyme, put into 37 DEG C of thermostat water bath water bath processing;
C, in liquid nitrogen cooling after, in boiling water bath place process, so repeatedly;
D, add SDS(neopelex) and Proteinase K, put into 65 DEG C of thermostat water baths and digest, after being down to normal temperature, carry out centrifugal treating;
E, get centrifugal in step D after the supernatant 8mol/L KAc that adds precooling carry out ice bath after, carry out centrifugal treating;
F, get step e centrifugal after supernatant add phenol chloroform isoamyl alcohol, carry out centrifugal treating;
G, get step F centrifugal after supernatant add chloroform isoamyl alcohol, carry out centrifugal treating;
H, get step G centrifugal after supernatant add 3mol/L NaAc and pre-cold isopropanol, precipitation at room temperature 2h;
I, carry out centrifugal treating after, discard centrifugal after supernatant, with ethanol washing precipitation 3 times, naturally dry, adding TE(TE is Tris-EDTA buffer solution, market buy, constituent: 10mmol/L Tris-HCl, 1mmol/LEDTA, 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 buffer solution, vortex oscillation 5min, the centrifugal 5min of 6000r/min, abandons supernatant, then adds 1mlTE1 buffer solution, the centrifugal 5min of vortex oscillation 5min, 6000r/min, abandons supernatant;
B, add 600 μ L TE2 Extraction buffers and 10 μ L concentration are the lysozyme of 100mg/mL, put into thermostat water bath 37 DEG C of water-bath 1h, every 10min puts upside down mixing once;
C, in liquid nitrogen, cool 5min, in boiling water bath, place 5min rapidly, so repeat 3 times;
D, add 120 μ L20%SDS and 10 μ L100mg/mL Proteinase Ks, put into thermostat water bath 65 DEG C digestion 1h, period, every 10min put upside down mixing once, after being down to normal temperature, and the centrifugal 10min of 8000r/min;
E, get centrifugal in step D after supernatant add the 8mol/L KAc of the precooling of 0.2 times of volume, ice bath 20min, 10000r/min4 DEG C of centrifugal 10min;
F, get step e centrifugal after supernatant add isopyknic phenol chloroform isoamyl alcohol (phenol: chloroform: isoamyl alcohol volume ratio is 25:24:1), the centrifugal 10min of 12000r/min;
G, get step F centrifugal after supernatant add isopyknic chloroform isoamyl alcohol (chloroform: isoamyl alcohol volume ratio is 24:1), the centrifugal 10min of 12000r/min;
H, get step G centrifugal after supernatant add the pre-cold isopropanol of 3mol/L NaAc and the 0.6 times volume of 0.1 times of volume, precipitation at room temperature 2h;
I, 14000r/min4 DEG C of centrifugal 10min, discard centrifugal after supernatant, be the ethanol washing precipitation 3 times of 70% by 500 μ L concentration, naturally dry, add 50 μ LTE dissolution precipitations.
Described TE1 buffer formulation: 0.1mol/L phosphate buffer, 3mol/L EDTA, 0.1mol/L Tris-HCl, pH=8.0:
Described TE2 Extraction buffer formula: 0.1mol/L phosphate 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, in acid, 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 lysozyme, obtain the genome DNA of high concentration, first need removal heavy metal, regulate electrical conductivity and pH, avoid it on the impact of lysozyme activity.EDTA can chelate heavy metals effectively, uses the buffer solution with high concentration EDTA can remove heavy metals a large amount of in mine tailing before extracting mine tailing heavy metal.Use buffer solution of the present invention effectively can stablize pH and the electrical conductivity of sample.Simultaneously for ensureing the purity of genome DNA, Proteinase K, SDS and phenol chloroform isoamyl alcohol (phenol: chloroform: isoamyl alcohol volume ratio is 25 ﹕ 24 ﹕ 1) can remove a large amount of protein, and CTAB, KAc also can Polysaccharide removing class materials effectively.The present invention is simple to operate, and the DNA band of acquisition is clear, and integrality is good, and purity is high, and impurity content is few.
Specific embodiment:
Embodiment one, extract acidifying heavy metal mine tailing genome DNA, comprise step:
(1) get about 0.5g acidifying heavy metal mine tailing, be placed in 1.5mL centrifuge tube, add 1mL TE1 buffer solution, vortex oscillation 5min, the centrifugal 5min of 6000r/min, abandons supernatant, then adds 1mlTE1 buffer solution, the centrifugal 5min of vortex oscillation 5min, 6000r/min, abandons supernatant.
(2) add 600 μ L TE2 Extraction buffers and thermostat water bath 37 DEG C of water-bath 1h put into by 10 μ L lysozymes (100mg/mL), every 10min puts upside down mixing once.
(3) in liquid nitrogen, cool 5min, in boiling water bath, put into 5min rapidly, so repeat 3 times.
(4) add 120 μ L20%SDS and 10 μ L100mg/mL Proteinase Ks, put into thermostat water bath 65 DEG C digestion 1h, period, every 10min put upside down mixing once.Be down to normal temperature, the centrifugal 10min of 8000r/min.
(5) get above-mentioned centrifugal after supernatant add the 8mol/L KAc of the precooling of 0.2 times of volume, ice bath 20min, 10000r/min4 DEG C of centrifugal 10min.
(6) get above-mentioned centrifugal after supernatant add isopyknic phenol chloroform isoamyl alcohol (phenol: chloroform: isoamyl alcohol volume ratio is 25 ﹕ 24 ﹕ 1), the centrifugal 10min of 12000r/min.
(7) get above-mentioned centrifugal after supernatant add isopyknic chloroform isoamyl alcohol (chloroform: isoamyl alcohol volume ratio is 24:1), the centrifugal 10min of 12000r/min.
(8) get above-mentioned centrifugal after supernatant add the pre-cold isopropanol of 3mol/L NaAc and the 0.6 times volume of 0.1 times of volume, precipitation at room temperature 2h.
(9) 14000r/min4 DEG C of centrifugal 10min, discard above-mentioned centrifugal after supernatant, with 500 μ L70% ethanol washing precipitation 3 times, naturally dry, add 50 μ LTE dissolution precipitations.
Get 3 μ L to detect for 0.8% agarose gel electrophoresis, as shown in Figure 1, show that the DNA brightness obtained by the inventive method is high, abundanter, more clear, the repeatability of sample room is better for result.
Compared with soil DNA extracting method traditional in prior art, the DNA that this method is extracted has better stability, and the DNA concentration extracted is higher.
Embodiment two, the concentration of genome DNA and purity in working sample:
Utilize concentration and the purity of UV spectrophotometer measuring DNA, survey the light absorption value of DNA solution when 260nm, 280nm of extraction respectively, the light absorption value that the inventive method extracts 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.
The purity of the acidifying heavy metal mine tailing genome DNA that table 1 the inventive method is extracted and concentration
The purity of the acidifying heavy metal mine tailing genome DNA that soil DNA extracting method traditional in table 2 prior art extracts and concentration
Conclusion: compared with soil DNA extracting method traditional in prior art, the DNA purity that the inventive method is extracted is higher, and concentration is comparatively large, and effect is very desirable.
Embodiment three, the PCR primer of the 16S rRNA of acidifying heavy metal mine tailing genome DNA detects:
Use bacterial universal primers 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+, Taq DNA polymerase), DNA profiling 2 μ L, each 2 μ L of primer, sterilizing distilled water 19 μ L.PCR reaction system: 94 DEG C of sex change 60s, 50 DEG C of annealing 30s, 72 DEG C extend 60s, 35 circulations.Get 5 μ L products electrophoresis detection in 0.8% Ago-Gel.
Result: as shown in Figure 3, sample all realizes effective amplification, and amplified production band is clear, and fragment is about about 1500bp, and specific fragment length is consistent, illustrates that the genome DNA in the acidifying heavy metal mine tailing that this method is extracted can meet follow-up PCR experiment etc.
The foregoing is only the present invention's preferably detailed description of the invention; but protection scope of the present invention is not limited thereto; anyly be familiar with those skilled in the art in the technical scope that the present invention discloses, the change that can expect easily or replacement, all should be encompassed within 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 (1)
1. extract a method for genome DNA in acidifying heavy metal mine tailing, it is characterized in that, comprise step:
A, get 0.5g acidifying heavy metal mine tailing, be placed in 1.5mL centrifuge tube, add 1mL TE1 buffer solution, vortex oscillation 5min, the centrifugal 5min of 6000r/min, abandons supernatant, then adds 1mL TE1 buffer solution, the centrifugal 5min of vortex oscillation 5min, 6000r/min, abandons supernatant;
B, add 600 μ L TE2 Extraction buffers and 10 μ L concentration are the lysozyme of 100mg/ml, put into thermostat water bath 37 DEG C of water-bath 1h, every 10min puts upside down mixing once;
C, in liquid nitrogen, cool 5min, in boiling water bath, place 5min rapidly, so repeat 3 times;
D, add 120 μ L 20%SDS and 10 μ L 100mg/mL Proteinase Ks, put into thermostat water bath 65 DEG C digestion 1h, period, every 10min put upside down mixing once, after being down to normal temperature, and the centrifugal 10min of 8000r/min;
E, get centrifugal in step D after supernatant add the 8mol/L KAc of the precooling of 0.2 times of volume, ice bath 20min, 10000r/min 4 DEG C of centrifugal 10min;
F, get step e centrifugal after supernatant add isopyknic phenol: chloroform: isoamyl alcohol volume ratio is the phenol chloroform isoamyl alcohol of 25 ﹕ 24 ﹕ 1, the centrifugal 10min of 12000r/min;
G, get step F centrifugal after supernatant add isopyknic chloroform: isoamyl alcohol volume ratio is the chloroform isoamyl alcohol of 24 ﹕ 1, the centrifugal 10min of 12000r/min;
H, get step G centrifugal after supernatant add the pre-cold isopropanol of 3mol/L NaAc and the 0.6 times volume of 0.1 times of volume, precipitation at room temperature 2h;
I, 14000r/min 4 DEG C of centrifugal 10min, discard centrifugal after supernatant, be the ethanol washing precipitation 3 times of 70% by 500 μ L concentration, naturally dry, add 50 μ LTE dissolution precipitations;
Described TE1 buffer formulation: 0.1mol/L phosphate buffer, 3mol/L EDTA, 0.1mol/L Tris-HCl, pH=8.0:
Described TE2 Extraction buffer formula: 0.1mol/L phosphate buffer, 3mol/L EDTA, 0.1mol/L Tris-HCl, 1.5mol/L NaCl, 1%CTAB, pH=8.0.
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| 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 |
| 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 |
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| 一种直接用于PCR 分析的风沙土微生物总DNA 提取方法;张颖等;《东北大学学报（自然科学版）》;20101130;第31卷(第11期);第1641页第1.2节 * |
| 提取北方土壤真菌DNA的一种方法;吴敏娜等;《生态学杂志》;20070430;第26卷(第4期);第611-616页 * |
| 磁铁矿尾矿宏基因组DNA提取方法的初步研究;吕志堂等;《安徽农业科学》;20100430;第38卷(第7期);第3361页第1.2.1.1节 * |
| 风沙土微生物总DNA不同提取和纯化方法比较;徐晓皎等;《应用生态学报》;20100531;第21卷(第5期);第1328页第1.3.1节 * |
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