CN1990864A - Extraction method of acidophilic bacteria genome DNA in wet method metallurgy - Google Patents
Extraction method of acidophilic bacteria genome DNA in wet method metallurgy Download PDFInfo
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- CN1990864A CN1990864A CNA2005101355384A CN200510135538A CN1990864A CN 1990864 A CN1990864 A CN 1990864A CN A2005101355384 A CNA2005101355384 A CN A2005101355384A CN 200510135538 A CN200510135538 A CN 200510135538A CN 1990864 A CN1990864 A CN 1990864A
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- genome dna
- acidophilic bacteria
- hydrometallurgy
- dna
- bacteria genome
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P10/00—Technologies related to metal processing
- Y02P10/20—Recycling
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Abstract
The invention provides a novel, fast and stable method for extracting genome DNA from pulp acidophilic bacteria. It comprises following steps: centrifuging cultured pulp, washing with 0.2 M sulfate and SET buffer solution respectively; adding lysozyme and treating for 30 minutes at 37 Deg. C, adding protein enzyme K and SDS and treating for 45 minutes at 50 Deg. C, adding glass balls and vortexing for 1 minute to crack cell completely, then extrcting protein with chloroform, washing with waterless alcohol precipitation and alcohol of 75% and getting pure genome DNA, which can be used for PCR gene operation.
Description
Technical field
The present invention relates to the extracting method of acidophilic bacteria genome DNA.
Technical background
Utilize acidophilic bacteria that the method for metal stripping in lean ore and the mine tailing and recovery is called biological hydrometallurgy (biohydrometallurgy).As far back as 18th century, in Spain Rio Tinto mine, people just know from water is leached in the mine and reclaim copper.After separating and identifying thiobacillus ferrooxidant, the 1950's, biological hydrometallurgy formally was identified as a technology.The U.S. began to utilize biological hydrometallurgical extraction copper in 1958, and Canada began to extract uranium in 1966, and then wet method is extracted metals such as zinc, cobalt, nickel, gold and also obtained success, and biological hydrometallurgy is widely used in the whole world.1997 and calendar year 2001, China also builds up two biologies more than the kiloton at Jiangxi Dexing Copper Mine and the Purple Mountain, Fujian copper mine respectively and carries copper dump leaching factory.Recent two decades comes, and biological oxidation process extracts the golden hair exhibition rapidly.At present, in South Africa, country such as Australia, Brazil, Ghana, Canada and Zimbabwe built up nearly 20 tame bacterial oxidation gold extracting shops.Though the application of biological oxidation gold extraction technology on China's gold industry started late,, develop very rapid because it tallies with the national condition.After 2000, China respectively in Yantai, Shandong, Laizhou and Fengcheng City, Liaoning build up three bacterial oxidation gold extracting shops.
Biological hydrometallurgy is compared with other method, reaction temperature and, environmental friendliness, energy consumption is low, flow process is short.In ore poor day by day assorted and today that environmental problem is outstanding, biological hydrometallurgical technology will be that effective metallic element extracts and the means of utilization of waste material.
At present, the application of biological hydrometallurgy in industrial production is increasingly extensive, and this effectively monitors the acidophilic bacteria in the hydrometallurgical processes with regard to needing people, with control reaction conditions, optimization reaction parameter.But because the acidophilic bacteria individuality is small, so be difficult to general observation by light microscope monitoring, other Physiology and biochemistry method is very loaded down with trivial details again, wastes time and energy.Protocols in Molecular Biology develop into hydrometallurgy produce in the acidophilic bacteria monitoring effective means are provided.Recently, existing abroad investigator utilizes the development and change of acidophilic bacteria group in the research hydrometallurgys such as gene clone library technology, SSCP, 16S-23S intergenic sequence technology.These technology all are based on acidophilic bacteria genome DNA.At present, though be used to separate and to extract the method for microbe genome DNA a lot, and also comparative maturity all.But the acidophilic bacteria in the hydrometallurgy is to be present in reactor pH to be lower than in 2.0 the ore pulp, and existence is very special, all is not suitable for acidophilic bacteria so be used to extract the method for other microbe genome DNA.The acidophilic bacteria DNA extraction method formality of reporting in the pertinent literature is loaded down with trivial details, and the result is unsatisfactory.Therefore, this just needs us to seek a kind of acidophilic bacteria DNA extraction method of simple and convenient and effect stability.
Summary of the invention
The objective of the invention is for a kind of new, extracting method that is applicable to ore pulp acidophilic bacteria genome DNA simple and fast, effect stability is provided.
The extracting method of described microbe genome DNA, it may further comprise the steps:
1, microorganism collection: 10000rpm collected cultured ore pulp in centrifugal 8 minutes, abandoned supernatant.
2, thalline washing: with 0.02M sulfuric acid scrubbing ore pulp twice, to remove to the deleterious heavy metal ion of follow-up work; Again with STE buffered soln washing one time (sucrose 10%, TrisHCl 50mM, EDTA 10mM, NaCl 100mM pH8.0), transfers to 8.0 with slurry pH, in order to avoid the DNA that sour environment hydrolysis subsequent step discharges.
3, cellular lysate: with STE buffered soln suspension ore pulp, add N,O-Diacetylmuramidase, 37 ℃ of water bath processing 30 minutes to final concentration 5mg/ml; Add Proteinase K to final concentration 3mg/ml, SDS was to 2%, 50 ℃ of water bath processing of final concentration 45 minutes; Adding diameter is the granulated glass sphere 0.5g of 0.1mm, and low-speed oscillation is 1 minute on the vortex oscillation device.
4, remove albumen:, get supernatant with centrifugal 8 minutes of the solution 10000rpm after the cracking; Add isopyknic chloroform extracting twice, get supernatant.
5, the precipitation of genomic dna: the 3M sodium acetate of adding 10% in supernatant liquor, add isopyknic Virahol again, placed 1 hour for-20 ℃; Centrifugal 10 minutes of 12000rpm abandons supernatant.
6, precipitation with 75% washing with alcohol once, seasoning is dissolved with 50 μ L sterilized waters at last, and to add 1 μ L concentration be the RNase of 1 μ g/mL, promptly obtains acidophilic bacteria genome DNA purer, that can be directly used in PCR equimolecular biologic operation.
Description of drawings
Accompanying drawing is the agarose electrophoresis collection of illustrative plates of DNA, and 1 swimming lane wherein is the electrophoretogram of lambda bacteriophage dna behind HindIII restricted type endonuclease digestion; Two swimming lanes on the left side are respectively the design sketch of the extracting genome DNA of following three embodiment.
Below describe working of an invention in detail by concrete practical work example, purpose is to help the reader to understand essence of the present invention better, but not as the qualification to the scope of the present invention.
Embodiment 1:
Add the sulphur powder in the leathen substratum to final concentration 2%, regulate pH to 2.0, inoculation acidophilic bacteria 10%, 170rpm, 40 ℃ of constant-temperature shaking culture 48 hours with sulfuric acid.Centrifugal collection thalline, the extraction of its genomic dna the results are shown in accompanying drawing the 2nd swimming lane, and the output of extracting DNA is 35 μ g/g ore pulps.
Embodiment 2:
Get certain high arsenic difficulty and soak gold concentrate, with 9K substratum modulation ore pulp, pulp density 15%, pH1.8, inoculation acidophilic bacteria 10%, 170rpm, 40 ℃ of constant-temperature shaking culture 48 hours.Centrifugal collection thalline, the extraction of its genomic dna the results are shown in accompanying drawing the 3rd swimming lane, and the output of extracting DNA is 45 μ g/g ore pulps.
Embodiment 3
Get certain iron and dodge zinc ore concentrate, with 9K substratum modulation ore pulp, pulp density 10%, pH2.0, inoculation acidophilic bacteria 10%, 170rpm, 40 ℃ of constant-temperature shaking culture 48 hours.Centrifugal collection thalline, the extraction of its genomic dna the results are shown in accompanying drawing the 3rd swimming lane, and the output of extracting DNA is 50 μ g/g ore pulps.
Use A
260/ A
280Quality to three kinds of genomic dnas of above extraction detects, and ratio has all reached 1.8, and the result shows that the purity of three kinds of DNA is higher.
Claims (4)
1. the extracting method of acidophilic bacteria genome DNA in the hydrometallurgy is characterized in that the good ore pulp of centrifugal collection needs earlier to remove heavy metal ion with the sulfuric acid scrubbing twice of 0.2M; Again with STE buffered soln washing one time, the pH of ore pulp is transferred to 8.0.
2. the extracting method of acidophilic bacteria genome DNA in the described a kind of hydrometallurgy of claim 1 is characterized in that the composition of STE extraction buffered soln is: sucrose 10%, TrisHCl 50mM, EDTA 10mM, NaCl 100mM, pH8.0.
3. the extracting method of acidophilic bacteria genome DNA in the described a kind of hydrometallurgy of claim 1 is characterized in that the cellular lysate final step added the granulated glass sphere low-speed oscillation 1 minute, made the complete cracking of thalline.
4. the extracting method of acidophilic bacteria genome DNA in the described a kind of hydrometallurgy of claim 1 is characterized in that DNA need add 10% sodium acetate when precipitating.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2005101355384A CN1990864A (en) | 2005-12-30 | 2005-12-30 | Extraction method of acidophilic bacteria genome DNA in wet method metallurgy |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2005101355384A CN1990864A (en) | 2005-12-30 | 2005-12-30 | Extraction method of acidophilic bacteria genome DNA in wet method metallurgy |
Publications (1)
| Publication Number | Publication Date |
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| CN1990864A true CN1990864A (en) | 2007-07-04 |
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| CNA2005101355384A Pending CN1990864A (en) | 2005-12-30 | 2005-12-30 | Extraction method of acidophilic bacteria genome DNA in wet method metallurgy |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102161987A (en) * | 2010-02-20 | 2011-08-24 | 大连水产学院 | Method for extracting genomic deoxyribonucleic aid (DNA) of sediment microorganisms in mariculture pond |
| RU2648158C2 (en) * | 2016-08-15 | 2018-03-22 | Федеральное государственное учреждение Федеральный исследовательский центр "Фундаментальные основы биотехнологии" Российской академии наук | Method of preparation of mine waters for dna selection |
-
2005
- 2005-12-30 CN CNA2005101355384A patent/CN1990864A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102161987A (en) * | 2010-02-20 | 2011-08-24 | 大连水产学院 | Method for extracting genomic deoxyribonucleic aid (DNA) of sediment microorganisms in mariculture pond |
| CN102161987B (en) * | 2010-02-20 | 2013-03-27 | 大连水产学院 | Method for extracting genomic deoxyribonucleic aid (DNA) of sediment microorganisms in mariculture pond |
| RU2648158C2 (en) * | 2016-08-15 | 2018-03-22 | Федеральное государственное учреждение Федеральный исследовательский центр "Фундаментальные основы биотехнологии" Российской академии наук | Method of preparation of mine waters for dna selection |
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