CN101215559A - Method for separating and purifying arthraspira cyanobacteria plasmid - Google Patents
Method for separating and purifying arthraspira cyanobacteria plasmid Download PDFInfo
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- CN101215559A CN101215559A CNA2008100590817A CN200810059081A CN101215559A CN 101215559 A CN101215559 A CN 101215559A CN A2008100590817 A CNA2008100590817 A CN A2008100590817A CN 200810059081 A CN200810059081 A CN 200810059081A CN 101215559 A CN101215559 A CN 101215559A
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Abstract
The invention relates to a method for separating and purifying arthrospira cyanobacteria plasmids. Conventional CTAB method or SDS method also can extract out clear genome DNA, but plasmids can not be extracted out by the SDS method, the plasmids which are extracted by the CTAB method are vaguely visible and blurring, which can not satisfy the requirements for research. The CTAB-proteinaseK method of the invention which comprises adding suitable proteinase-K into CTAB extract and DNA solution after the first deposition not only can extract the clear genome DNA but also can obtain clear plasmids, furthermore, the plasmids with high quality and integrity can be obtained through using the gel freezing thawing- high speed centrifugal purification method of the invention, which comprises following steps: cutting a gel belt which contains the plasmids into a sterilizing centrifuge tube, adding TE buffering liquid whose volume is same as a gel block, freezing 60 minutes at minus 20 DEG C, then, taking out, melting, transferring into a micropore centrifuge column which is provided with sterilizing absorbent cotton or glass wool, and centrifuging for 15 minutes with 13000rpm per minute, the plasmids are in solution on the bottom of the centrifuge tube, and integral plasmids with high quality can be obtained.
Description
Technical field
The invention belongs to the technology of arthraspira cyanobacteria isolation and purification
Background technology
Arthraspira cyanobacteria (Arthrospira cyanobacteria) be a kind of by a plurality of cells be concatenated into thread not branch, be regular scroll, photosynthetic oxygen evolution, ancient prokaryotic micro-organisms, be not only the ideal material of great biology subject studies such as carrying out the origin of species and evolution, morphogenesis, photosynthesis, degeneration-resistant mechanism, and multiple nutrients such as, training fast because of its growth grown that facility is easy, production cost is low, edible safety, rich in proteins and polysaccharide and biologically active substance are large-scale developed and utilized in the whole world.Grow production technology, nutritive health-care, pharmacology etc. with Physiology and biochemistry, training and compare, the research relative deficiency of the molecular genetics aspect of relevant arthraspira cyanobacteria is having a strong impact on the further investigation and the development and use of current arthraspira cyanobacteria.DNA is biological genetic material, and research also discloses the genetic information of DNA, and structure-function relationship etc. is significant to fundamental research and the Application and Development of pushing forward arthraspira cyanobacteria comprehensively.Biological the same with other, except that genomic dna is arranged, also have some to be known as the small molecule DNA of plasmid in the arthraspira cyanobacteria, they in the form of a ring or wire are not only being undertaken important biological function, and are being the ideal elements of structure transgene carrier.
Setting up efficient, the high-quality isolation and purification method of plasmid, is the basis of carrying out follow-up research and development such as its sequence, structure, function, transgene carrier structure.(Oceanologia et Limnologia Sinica such as Qin Song, 1994,560～563), (Plant Sci such as Lee 25 (5):, 1995,106:99～105), (Chin J Oceanol Limnol, 1998 such as Kojima, 16:30～39) separation and purification of plasmid in the cyanobacteria and characteristic etc. some researchs have been done, but, do not see the relevant report that solves this difficult problem both at home and abroad as yet because the protein and the polysaccharide content height of arthraspira cyanobacteria all are difficult to obtain high-quality plasmid with conventional extracting method such as SDS method or CTAB methods.
Summary of the invention
The present invention seeks to set up the method for a kind of easy, high efficiency separation and purifying high quality arthraspira cyanobacteria plasmid at prior art problems.
The present invention be the CTAB extracting solution and for the first time the DNA lysate of post precipitation be to add an amount of Proteinase K in the TE damping fluid; And then utilize gel freeze thawing-supercentrifugal process, and plasmid is made further efficiently concentrating and purifying, obtain the arthraspira cyanobacteria plasmid of high quality, structural integrity with this.
We utilize conventional CTAB method and SDS method to extract total DNA of arthraspira cyanobacteria strain Ap-2mz respectively and find through electrophoretic analysis, with two kinds of methods can both carry genomic dna clearly, but carry less than plasmid with the SDS method, and can only mention the plasmid that mays be seen indistinctly, blurs with the CTAB method, can not satisfy further institute needs (Fig. 1).On this basis, we organically combine CTAB method and enzyme process, set up the CTAB-Proteinase K method of arthraspira cyanobacteria DNA extraction first, on electrophorogram, can not only see genomic dna clearly, and also had the plasmid band (Fig. 1) of clear, an about 0.75kb of molecular weight.Pass through VersaDoc
TMImaging System 3000 gel imaging system (Bio-Rad, USA) Quantity One software analysis shows, at arthraspira cyanobacteria lysate CTAB and for the first time add an amount of Proteinase K in the DNA lysate of post precipitation, the extraction yield of plasmid can be improved nearly 3 times, now need thereby electrophoretic band is clear, can satisfy follow-up study.
Simultaneously, the follow-up study needs such as order-checking, structure and function for satisfying arthraspira cyanobacteria plasmid are necessary it is made further enrichment and purifying.We have tried out some purifying commonly used and have reclaimed test kit, but the purity and the rate of recovery etc. are all undesirable.Through repetition test and improvement, we have set up a kind of method that the gel freeze thawing is combined with technology such as high speed centrifugations, i.e. gel freeze thawing-high speed centrifugation method of purification.With the plasmid (Fig. 1) of this method purifying, electrophoretic band single illustrates the purity height; Shape and position do not change, and illustrate that molecular structure is complete; With gel imaging system plasmid electrophoretic band before and after the purifying is shown that do check and analysis organic efficiency is up to more than 90%.
Remarkable advantage of the present invention:
The arthraspira cyanobacteria plasmid isolation and purification method that the present invention set up is compared with ordinary method, has easy and simple to handle, characteristics such as yield is high, quality good, structural integrity.
Description of drawings
Fig. 1 be utilize Different Extraction Method carry the electrophorogram of the total DNA of arthraspira cyanobacteria strain Ap-2mz, wherein M is a standard molecular weight: 1 expression CTAB-Proteinase K method; 2 expression CTAB methods; 3 expression SDS methods;
Fig. 2 is the electrophorogram of the plasmid of purifying from arthraspira cyanobacteria strain Ap-2mz; Wherein M is a standard molecular weight: the plasmid of 1 expression purifying;
Fig. 3 is the electrophorogram that utilizes the plasmid of total DNA that CTAB-Proteinase K method extracts and purifying from tested arthraspira cyanobacteria strain Ap-JS10m, and wherein M is a standard molecular weight: the total DNA of 1 expression; The plasmid of 2 expression purifying.
Embodiment
Detailed technology scheme of the present invention can be implemented by following steps:
1, select materials is known arthraspira cyanobacteria strain Ap-2mz, Ap-JS10m (also there is preservation in Zhejiang University's nucleus research of agricultural science institute's Biological resources and molecular engineering laboratory).
2, reagent and instrument
Agarose, CTAB (cetyl trimethylammonium bromide), SDS (sodium laurylsulfonate), the centrifugal post of micropore (SK131/132 post) are given birth to worker bio-engineering corporation product for Shanghai; Proteinase K is a Merck company product; RNaseA is Japanese TaKaRa company product; Other reagent is analytical pure.The gel electrophoresis analysis system is a U.S. Amersham company product; The gel imaging analysis system is a U.S. Bio-Rad company product.
3, the separation and Extraction of arthraspira cyanobacteria plasmid
(1) gets the algae mud (or about 15mg of dry algae powder of stored frozen) of fresh algae mud of about 250mg or stored frozen in the 5ml centrifuge tube of sterilization, earlier with liquid nitrogen to sample freeze thawing treatment 3～5 times after, add 2ml and be preheated to 65 ℃ CTAB extracting solution [2%CTAB, 50mmol/L Tris-HCl (pH8.0), 10mmol/L EDTA, 0.7mol/L NaCl, 1% (V/V) beta-mercaptoethanol (adding before using)], and add an amount of Proteinase K solution to final concentration 300 μ g/ml; Again sample is put upside down and is mixed, in 62 ℃ of water-bath 1h, during every about 10min, sample is shaken up once gently.
(2) under the room temperature 10,000rpm is centrifugal, and 5min removes impurity such as cell debris, and supernatant liquor adds equal-volume chloroform/primary isoamyl alcohol (24: 1), puts upside down behind the mixing in 12 the centrifugal 10min of 000rpm gently repeatedly.
(3) get supernatant liquor in another sterilization centrifuge tube, add-20 ℃ of precooling dehydrated alcohols of 3 times of volumes and the 3mol/LNaAc (pH4.8) of 1/10 volume, put upside down for several times the back gently and place 1h down in-20 ℃, DNA is fully precipitated.
The centrifugal 10min of (4) 12,000rpm.For the first time post precipitation DNA is with 70% washing with alcohol, and vacuum is drained, and is dissolved in 500 μ l TE-Proteinase K damping fluid (10mmol/L Tris-HCl, 1mmol/L EDTA adds Proteinase K to final concentration 200 μ g/ml, pH8.0), also shake gently frequently in 50 ℃ of water-bath 1h, clarify until solution.
(5) add isopyknic phenol/chloroform/primary isoamyl alcohol (25: 24: 1), put upside down mixing gently, 12, the centrifugal 10min of 000rpm.
(6) get supernatant liquor, add the 3mol/LNaAc of 1/10 volume and the dehydrated alcohol of 3 times of volume-20 ℃ precoolings, place 1h in-20 ℃.
(7) 12,4 ℃ of centrifugal 10min of 000rpm, for the second time post precipitation DNA is with 70% washing with alcohol 2 times, vacuum is drained, and adds 100 μ l TE-RNase damping fluid (10mmol/L Tris-HCl, 1mmol/L EDTA, 50 μ g/ml RNase, pH8.0), in 37 ℃ of water-baths, be incubated 30min, make the DNA dissolving.
4, electrophoretic analysis and observation
With carry DNA electrophoresis on 1% sepharose, voltage 4V/cm.Thereafter with the about 30min of EB dyeing, again in VersaDocTM Imaging System 3000 gel imaging analysis systems (Bio-Rad, USA) observation and photograph.
5, the method for purification of arthraspira cyanobacteria plasmid
(1) cuts off the sepharose piece at plasmid place in the ultraviolet lamp long-wave band, put into the sterilization centrifuge tube, add the TE damping fluid of 1 times of blob of viscose volume approximately ,-20 ℃ of freezing 60min.
(2) take out the refrigerated centrifuge tube, treat that glue melts after, glue is transferred in the centrifugal post of micropore that is covered with one deck sterilization absorbent cotton (or glass wool) with solution, 13, the centrifugal 15min of 000rpm is in the solution that plasmid is promptly collected in centrifuge tube.
(3) in reclaiming liquid, add equal-volume phenol/chloroform/primary isoamyl alcohol, behind the mixing under room temperature 12, the centrifugal 5min of 000rpm.
(4) get supernatant, add 0.1 times of volume 3mol/L NaAc and 2.5 times of volume precooling dehydrated alcohols, place 30min for-20 ℃, 12, the centrifugal 10min of 000rpm, precipitation is with 70% washing with alcohol 2 times, and vacuum is drained, and adds 20 μ l distilled waters and dissolves, in-20 ℃ of preservations.
(5) electrophoretic analysis and observation.Method is with described in 4.
Result and analysis
Discover, though conventional CTAB method and SDS method can carry the genomic dna of arthraspira cyanobacteria clearly, all can not carry high-quality plasmid (Fig. 1).We slightly are better than the SDS method according to the CTAB method, and it is combined with enzyme process, set up the CTAB-Proteinase K method of arthraspira cyanobacteria DNA extraction first, promptly at arthraspira cyanobacteria lysate CTAB and for the first time add an amount of Proteinase K in the DNA lysate of post precipitation, then can carry in the high quality genomic dna, obtain high-quality plasmid (Fig. 1).The key takeaway of this method is to utilize amounts of protein in the Proteinase K enzymolysis arthraspira cyanobacteria, thereby makes DNA and protein dissociation and separated.Simultaneously, the gel freeze thawing-high speed centrifugation method of purification of arthraspira cyanobacteria plasmid that we set up, the purity of gained plasmid and rate of recovery height, structural integrity (Fig. 2).
As an example, we have chosen arthraspira cyanobacteria strain Ap-JS10m and have verified practicality of the present invention.Utilize CTAB-Proteinase K method to extract its total DNA and do electrophoretic analysis, the plasmid that as shown in Figure 3, utilize that institute's construction method can be carried clearly, high-quality genomic dna and molecular weight is about 1300bp.Further use the gel freeze thawing-high speed centrifugation method of purification of being built, obtained single, the plasmid clearly of band.In addition, from result of study as can be known, the plasmid that different strain segmentum intercalaris revolve cyanobacteria is not quite similar, and this has also embodied the necessity of its species diversity and research.
Claims (2)
1. the isolation and purification method of an arthraspira cyanobacteria plasmid, it is characterized in that the CTAB extracting solution and for the first time the DNA lysate of post precipitation be to add an amount of Proteinase K in the TE damping fluid; And then utilize gel freeze thawing-supercentrifugal process, and plasmid is made further efficiently concentrating and purifying, obtain the arthraspira cyanobacteria plasmid of high quality, structural integrity with this.
2. by the isolation and purification method of the described arthraspira cyanobacteria plasmid of claim 1, it is characterized in that adopting following operation steps:
(1) select materials is known arthraspira cyanobacteria strain Ap-2mz, Ap-JS10m;
(2) reagent and instrument: agarose, CTAB are that cetyl trimethylammonium bromide, SDS are that the centrifugal post of sodium laurylsulfonate, micropore is that the SK131/132 post is that worker bio-engineering corporation product is given birth in Shanghai, Proteinase K is a Merck company product, RNaseA is Japanese TaKaRa company product, other reagent is analytical pure, the gel electrophoresis analysis system is a U.S. Amersham company product, and the gel imaging analysis system is a U.S. Bio-Rad company product;
(3) separation and Extraction of arthraspira cyanobacteria plasmid
The about 15mg of dry algae powder that gets the algae mud of fresh algae mud of about 250mg or stored frozen or stored frozen is in the 5ml centrifuge tube of sterilization, earlier with liquid nitrogen to sample freeze thawing treatment 3～5 times after, adding 2ml is preheated to 65 ℃ CTAB extracting solution and comprises 2%CTAB, the 50mmol/L Tris-HCl of pH8.0,10mmol/L EDTA, 0.7mol/L NaCl, 1% (V/V) beta-mercaptoethanol adds before using, and add an amount of Proteinase K solution to final concentration 300 μ g/ml, again sample is put upside down and mixed, in 62 ℃ of water-bath 1h, during every about 10min, sample is shaken up once gently; Under the room temperature 10,000rpm is centrifugal, and 5min removes impurity such as cell debris, and supernatant liquor adds equal-volume chloroform/primary isoamyl alcohol (24: 1), puts upside down behind the mixing in 12 the centrifugal 10min of 000rpm gently repeatedly; Get supernatant liquor in another sterilization centrifuge tube, the 3mol/LNaAc that adds the pH4.8 of-20 ℃ of precooling dehydrated alcohols of 3 times of volumes and 1/10 volume puts upside down for several times the back gently and places 1h down in-20 ℃, and DNA is fully precipitated; 12, the centrifugal 10min of 000rpm.For the first time post precipitation DNA is with 70% washing with alcohol, and vacuum is drained, and being dissolved in 500 μ l TE-Proteinase K damping fluids is 10mmol/L Tris-HCl, 1mmol/L EDTA adds Proteinase K to final concentration 200 μ g/ml, pH8.0, also shake gently frequently in 50 ℃ of water-bath 1h, clarify until solution; Add isopyknic phenol/chloroform/primary isoamyl alcohol 25: 24: 1, and put upside down mixing gently, 12, the centrifugal 10min of 000rpm; Get supernatant liquor, add the 3mol/LNaAc of 1/10 volume and the dehydrated alcohol of 3 times of volume-20 ℃ precoolings, place 1h in-20 ℃; 12,4 ℃ of centrifugal 10min of 000rpm, for the second time post precipitation DNA is with 70% washing with alcohol 2 times, and vacuum is drained, adding 100 μ l TE-RNase damping fluids is 10mmol/LTris-HCl, 1mmol/L EDTA, 50 μ g/ml RNase, pH8.0 is incubated 30min in 37 ℃ of water-baths, make the DNA dissolving;
(4) electrophoretic analysis and observation
With carry DNA electrophoresis on 1% sepharose, voltage 4V/cm.Thereafter with the about 30min of EB dyeing, again at VersaDoc
TMImaging System 3000 gel imaging analysis systems (Bio-Rad, USA) observation and photograph;
(5) utilize the plasmid of gel freeze thawing-supercentrifugal process purifying arthraspira cyanobacteria
Cut off the sepharose piece at plasmid place in the ultraviolet lamp long-wave band, put into the sterilization centrifuge tube, add the TE damping fluid of 1 times of blob of viscose volume approximately ,-20 ℃ of freezing 60min; Take out the refrigerated centrifuge tube, treat that glue melts after, glue is transferred in the centrifugal post of micropore that is covered with one deck sterilization absorbent cotton or glass wool with solution, 13, the centrifugal 15min of 000rpm is in the solution that plasmid is promptly collected in centrifuge tube; In reclaiming liquid, add equal-volume phenol/chloroform/primary isoamyl alcohol, behind the mixing under room temperature 12, the centrifugal 5min of 000rpm; Get supernatant, add 0.1 times of volume 3mol/L NaAc and 2.5 times of volume precooling dehydrated alcohols, place 30min for-20 ℃, 12, the centrifugal 10min of 000rpm, precipitation is with 70% washing with alcohol 2 times, and vacuum is drained, and adds 20 μ l distilled waters and dissolves, in-20 ℃ of preservations; Carry out electrophoretic analysis and observation by (4) described method.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102807978A (en) * | 2012-04-17 | 2012-12-05 | 浙江省海洋开发研究院 | Alga ribonucleic acid (RNA) extractant and using method |
| CN103757093A (en) * | 2013-11-04 | 2014-04-30 | 中国热带农业科学院环境与植物保护研究所 | Quantitative detection method for FOC race 4 from soil |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102807978A (en) * | 2012-04-17 | 2012-12-05 | 浙江省海洋开发研究院 | Alga ribonucleic acid (RNA) extractant and using method |
| CN102807978B (en) * | 2012-04-17 | 2014-02-26 | 浙江省海洋开发研究院 | Alga ribonucleic acid (RNA) extractant and using method |
| CN103757093A (en) * | 2013-11-04 | 2014-04-30 | 中国热带农业科学院环境与植物保护研究所 | Quantitative detection method for FOC race 4 from soil |
| CN103757093B (en) * | 2013-11-04 | 2016-07-13 | 中国热带农业科学院环境与植物保护研究所 | The method of No. 4 microspecies of detection by quantitative banana blight bacteria from soil |
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