CN106350511A - Extraction method for polysaccharides-enriched microbial genome DNA - Google Patents
Extraction method for polysaccharides-enriched microbial genome DNA Download PDFInfo
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- CN106350511A CN106350511A CN201610984531.8A CN201610984531A CN106350511A CN 106350511 A CN106350511 A CN 106350511A CN 201610984531 A CN201610984531 A CN 201610984531A CN 106350511 A CN106350511 A CN 106350511A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
Abstract
The invention discloses an extraction method for polysaccharides-enriched microbial genome DNA. The method comprises the following steps: 1) adding a proper amount of thallus to nucleic acid separating buffer liquor and 2-mercaptoethanol, and uniformly mixing in a water bath; 2) taking out and centrifuging, and sucking out the supernatant; 3) adding PBS liquor, vibrating and centrifuging, sucking and abandoning the supernatant; and repeating for one time; 4) adding the PBS liquor, vibrating and uniformly mixing with hand, sucking and abandoning the supernatant and repeating for one time; 5) washing the supernatant, adding STES and vibrating and uniformly mixing, adding phenol chloroform and vibrating and uniformly mixing; 6) moving the supernatant into a new centrifugal tube, adding RNaseA and uniformly mixing, and then standing at room temperature; 7) adding magnetic beads and binding buffer, and vibrating and uniformly mixing; 8) combining in a blood blending instrument; 9) placing on a magnetic holder, and sucking and abandoning the supernatant after the supernatant is clarified; 10) adding ethyl alcohol, standing and then removing the ethyl alcohol; 11) centrifuging for a short time, washing the ethyl alcohol, adding sterilizing liquid, blowing upon and uniformly mixing; and 12) standing at room temperature until the supernatant is clarified, and moving the supernatant into a new centrifugal tube.
Description
Technical field
The present invention relates to a kind of extracting method of the microbial gene dna rich in polysaccharide it is adaptable to require strict to dna
Three generations's sequencing technologies, belong to biological technical field.
Background technology
In recent years, develop into researcher microorganisms genome due to Protocols in Molecular Biology and sequencing technologies
Sequence, synthesis mechanism of secondary metabolite etc. provide good basis, and the acquisition of microbial genome is then these works
The basis made.
Microorganism wall complicated component, rich in capsular polysaccharide, produces many saccharides and secondary metabolitess in incubation.
In microorganism, the wide variety of sugar, has mucopolysaccharide, acidic polysaccharose and neutral sugar etc., if these saccharides can not go in extraction process
It is easy to pollution dna, reduce the purity of dna except totally, these polysaccharide can suppress restriction enzyme, ligase and polymerase etc.
The activity of enzyme, has a strong impact on the quality of follow-up sequencing data.
With the high speed development of sequencing technologies, the smrt technology of pacific bioscience causes that scientific research personnel's is extensive
Concern.This sequencing is also based on the principle being sequenced in synthesis, and this technology employs zero-mode wave guide (zmw)
(zero level waveguide).The process of sequencing: the nucleotide being fluorescently labeled phosphoric acid group is chained with template on polymerase active site
Close (every kind of Deoxydization nucleotide is by the dye marker without color), be inspired fluorescence, after fluorescent pulse terminates, labeled
Phosphoric acid group is cut and discharges, and polymerase transfers to next position, and next Deoxydization nucleotide is connected to and starts on site
Release fluorescent pulse, carries out next circulation.This technology is especially high to the quality of dna and purity requirement, and such as dna degraded then causes
Data volume is few, and the quality of data is poor, and splicing effect is undesirable；Dna purity is not high, has the Substances Pollution such as sugar or salt can cause to gather
Synthase activity reduces even inactivation, causes the waste of experimental cost.
The genome being in the past directed to polysaccharide quasi-microorganism extracts, and can only be extracted using test kit, but test kit is expensive, passes through
Cross post and also have different degrees of interrupting, cause dna imperfect.
Content of the invention
The technical problem to be solved is to extract for the genome of existing polysaccharide quasi-microorganism existing
Problem and a kind of extracting method of the microbial gene dna rich in polysaccharide is provided, the method is simply easily operated, take short,
Cost is relatively low, and band is complete, the impurity pollution such as Polysaccharide removing that can be clean it is adaptable to various rich in polysaccharide microbial genome
Extraction.
The technical problem to be solved can be achieved through the following technical solutions:
A kind of extracting method of the microbial gene dna rich in polysaccharide, comprises the steps:
1) take appropriate thalline, add 1.5ml separate nucleic acid buffer and 20ul 2 mercapto ethanol, concussion mixes, and puts 90 DEG C
10min in water-bath, period overturns and mixes several times；
2) take out, 6000r/min centrifugation 10min in centrifuge, now it can be seen that a large amount of polysaccharide exists in solution；
3) gently suction out supernatant with pipettor, be careful not to be drawn onto polysaccharide；
4) add 1ml 1x pbs solution, 60hz on beveller, shake 2s, 10000rpm is centrifuged 2min, is softly inhaled with rifle
Abandon supernatant；
5) 4 are repeated) step 1 time；
6) add 1ml 1x pbs solution, shaken with handss and mix, 10000rpm is centrifuged 2min, abandon supernatant with soft suction of rifle；
7) repeat step 6) 1 time；
8) after cleaning supernatant, add 500ul stes, concussion mixes, and adds 500ul phenol chloroform, shakes 30s；
9) 12000rpm/min, is centrifuged 5min；
10) careful transfer supernatant, to new centrifuge tube, adds 5ul rnasea, and after mixing of turning upside down, room temperature is placed
5min；
11) 50ul magnetic bead, 500ul binding buffer are added, concussion mixes；
12) combine 5min on blood blending instrument；
13) it is placed on magnetic frame, after supernatant clarification, inhale and abandon supernatant；
14) add 1ml 80% ethanol, after standing 30s, remove ethanol；
15) after of short duration centrifugation, clean ethanol, add appropriate aquesterilisa, piping and druming mixes；
16) room temperature places 2min, is placed on magnetic frame, and after supernatant clarification, transfer supernatant is in new centrifuge tube.
The extracting method of the present invention is first by the cell wall rupture of microorganism, and does not destroy nucleus, discharges in Cytoplasm
Polysaccharide, discard supernatant after centrifugation, then repeatedly cleaned with pbs, with Polysaccharide removing, subsequently extract the dna in nucleus, exempt from
Go polysaccharide to the interference extracted and the pollution to nucleic acid, the dna band extracting is single whole, purity is high, and method is easy,
In operation, the time is short, and with low cost.
Brief description
Fig. 1 and Fig. 2 is the electrophoresis result figure being extracted using this experimental technique.
Fig. 3 is the electrophoresis result figure being extracted using conventional ctab method.
Fig. 4 is the electrophoresis result figure being extracted using qiagen tissue＆blood kit.
Illustrate: 1-3: for staphylococcus aureuses；4-5: pseudomonas aeruginosa；6-7: actinomycetes；8-9: Oryza sativa L.
Yellow sporangium；10-11: vibrio.
Specific embodiment
Embodiment 1
A kind of extracting method of the microbial gene dna rich in polysaccharide, comprises the steps:
1) take the thalline being rich in right amount the staphylococcus aureuses of polysaccharide, add 1.5ml separate nucleic acid buffer and
20ul 2 mercapto ethanol, concussion mixes, and puts 10min in 90 DEG C of water-baths, and period overturns and mixes several times；
2) take out, 6000r/min centrifugation 10min in centrifuge, now it can be seen that a large amount of polysaccharide exists in solution；
3) gently suction out supernatant with pipettor, be careful not to be drawn onto polysaccharide；
4) add 1ml 1x pbs solution, 60hz on beveller, shake 2s, 10000rpm is centrifuged 2min, is softly inhaled with rifle
Abandon supernatant；
5) 4 are repeated) step 1 time；
6) add 1ml 1x pbs solution, shaken with handss and mix, 10000rpm is centrifuged 2min, abandon supernatant with soft suction of rifle；
7) repeat step 6) 1 time；
8) after cleaning supernatant, add 500ul stes, concussion mixes, and adds 500ul phenol chloroform, shakes 30s；
9) 12000rpm/min, is centrifuged 5min；
10) careful transfer supernatant, to new centrifuge tube, adds 5ul rnasea, and after mixing of turning upside down, room temperature is placed
5min；
11) 50ul magnetic bead, 500ul binding buffer are added, concussion mixes；
12) combine 5min on blood blending instrument；
13) it is placed on magnetic frame, after supernatant clarification, inhale and abandon supernatant；
14) add 1ml 80% ethanol, after standing 30s, remove ethanol；
15) after of short duration centrifugation, clean ethanol, add appropriate aquesterilisa, piping and druming mixes；
16) room temperature places 2min, is placed on magnetic frame, and after supernatant clarification, transfer supernatant is in new centrifuge tube.
Embodiment 2
A kind of extracting method of the microbial gene dna rich in polysaccharide, comprises the steps:
1) take the thalline being rich in right amount the pseudomonas aeruginosa of polysaccharide, add 1.5ml separate nucleic acid buffer and
20ul 2 mercapto ethanol, concussion mixes, and puts 10min in 90 DEG C of water-baths, and period overturns and mixes several times；
2) take out, 6000r/min centrifugation 10min in centrifuge, now it can be seen that a large amount of polysaccharide exists in solution；
3) gently suction out supernatant with pipettor, be careful not to be drawn onto polysaccharide；
4) add 1ml 1x pbs solution, 60hz on beveller, shake 2s, 10000rpm is centrifuged 2min, is softly inhaled with rifle
Abandon supernatant；
5) 4 are repeated) step 1 time；
6) add 1ml 1x pbs solution, shaken with handss and mix, 10000rpm is centrifuged 2min, abandon supernatant with soft suction of rifle；
7) repeat step 6) 1 time；
8) after cleaning supernatant, add 500ul stes, concussion mixes, and adds 500ul phenol chloroform, shakes 30s；
9) 12000rpm/min, is centrifuged 5min；
10) careful transfer supernatant, to new centrifuge tube, adds 5ul rnasea, and after mixing of turning upside down, room temperature is placed
5min；
11) 50ul magnetic bead, 500ul binding buffer are added, concussion mixes；
12) combine 5min on blood blending instrument；
13) it is placed on magnetic frame, after supernatant clarification, inhale and abandon supernatant；
14) add 1ml 80% ethanol, after standing 30s, remove ethanol；
15) after of short duration centrifugation, clean ethanol, add appropriate aquesterilisa, piping and druming mixes；
16) room temperature places 2min, is placed on magnetic frame, and after supernatant clarification, transfer supernatant is in new centrifuge tube.
Embodiment 3
A kind of extracting method of the microbial gene dna rich in polysaccharide, comprises the steps:
1) take the actinomycetic thalline being rich in polysaccharide in right amount, add 1.5ml separate nucleic acid buffer and 20ul 2- sulfydryl
Ethanol, concussion mixes, and puts 10min in 90 DEG C of water-baths, and period overturns and mixes several times；
2) take out, 6000r/min centrifugation 10min in centrifuge, now it can be seen that a large amount of polysaccharide exists in solution；
3) gently suction out supernatant with pipettor, be careful not to be drawn onto polysaccharide；
4) add 1ml 1x pbs solution, 60hz on beveller, shake 2s, 10000rpm is centrifuged 2min, is softly inhaled with rifle
Abandon supernatant；
5) 4 are repeated) step 1 time；
6) add 1ml 1x pbs solution, shaken with handss and mix, 10000rpm is centrifuged 2min, abandon supernatant with soft suction of rifle；
7) repeat step 6) 1 time；
8) after cleaning supernatant, add 500ul stes, concussion mixes, and adds 500ul phenol chloroform, shakes 30s；
9) 12000rpm/min, is centrifuged 5min；
10) careful transfer supernatant, to new centrifuge tube, adds 5ul rnasea, and after mixing of turning upside down, room temperature is placed
5min；
11) 50ul magnetic bead, 500ul binding buffer are added, concussion mixes；
12) combine 5min on blood blending instrument；
13) it is placed on magnetic frame, after supernatant clarification, inhale and abandon supernatant；
14) add 1ml 80% ethanol, after standing 30s, remove ethanol；
15) after of short duration centrifugation, clean ethanol, add appropriate aquesterilisa, piping and druming mixes；
16) room temperature places 2min, is placed on magnetic frame, and after supernatant clarification, transfer supernatant is in new centrifuge tube.
Embodiment 4
A kind of extracting method of the microbial gene dna rich in polysaccharide, comprises the steps:
1) take the thalline of the Oryza sativa L. Huang sporangium being rich in polysaccharide in right amount, add 1.5ml separate nucleic acid buffer and 20ul
2 mercapto ethanol, concussion mixes, and puts 10min in 90 DEG C of water-baths, and period overturns and mixes several times；
2) take out, 6000r/min centrifugation 10min in centrifuge, now it can be seen that a large amount of polysaccharide exists in solution；
3) gently suction out supernatant with pipettor, be careful not to be drawn onto polysaccharide；
4) add 1ml 1x pbs solution, 60hz on beveller, shake 2s, 10000rpm is centrifuged 2min, is softly inhaled with rifle
Abandon supernatant；
5) 4 are repeated) step 1 time；
6) add 1ml 1x pbs solution, shaken with handss and mix, 10000rpm is centrifuged 2min, abandon supernatant with soft suction of rifle；
7) repeat step 6) 1 time；
8) after cleaning supernatant, add 500ul stes, concussion mixes, and adds 500ul phenol chloroform, shakes 30s；
9) 12000rpm/min, is centrifuged 5min；
10) careful transfer supernatant, to new centrifuge tube, adds 5ul rnasea, and after mixing of turning upside down, room temperature is placed
5min；
11) 50ul magnetic bead, 500ul binding buffer are added, concussion mixes；
12) combine 5min on blood blending instrument；
13) it is placed on magnetic frame, after supernatant clarification, inhale and abandon supernatant；
14) add 1ml 80% ethanol, after standing 30s, remove ethanol；
15) after of short duration centrifugation, clean ethanol, add appropriate aquesterilisa, piping and druming mixes；
16) room temperature places 2min, is placed on magnetic frame, and after supernatant clarification, transfer supernatant is in new centrifuge tube.
Embodiment 5
A kind of extracting method of the microbial gene dna rich in polysaccharide, comprises the steps:
1) take the thalline being rich in the vibrio of polysaccharide in right amount, add 1.5ml separate nucleic acid buffer and 20ul2- sulfydryl second
Alcohol, concussion mixes, and puts 10min in 90 DEG C of water-baths, and period overturns and mixes several times；
2) take out, 6000r/min centrifugation 10min in centrifuge, now it can be seen that a large amount of polysaccharide exists in solution；
3) gently suction out supernatant with pipettor, be careful not to be drawn onto polysaccharide；
4) add 1ml 1x pbs solution, 60hz on beveller, shake 2s, 10000rpm is centrifuged 2min, is softly inhaled with rifle
Abandon supernatant；
5) 4 are repeated) step 1 time；
6) add 1ml 1x pbs solution, shaken with handss and mix, 10000rpm is centrifuged 2min, abandon supernatant with soft suction of rifle；
7) repeat step 6) 1 time；
8) after cleaning supernatant, add 500ul stes, concussion mixes, and adds 500ul phenol chloroform, shakes 30s；
9) 12000rpm/min, is centrifuged 5min；
10) careful transfer supernatant, to new centrifuge tube, adds 5ul rnasea, and after mixing of turning upside down, room temperature is placed
5min；
11) 50ul magnetic bead, 500ul binding buffer are added, concussion mixes；
12) combine 5min on blood blending instrument；
13) it is placed on magnetic frame, after supernatant clarification, inhale and abandon supernatant；
14) add 1ml 80% ethanol, after standing 30s, remove ethanol；
15) after of short duration centrifugation, clean ethanol, add appropriate aquesterilisa, piping and druming mixes；
16) room temperature places 2min, is placed on magnetic frame, and after supernatant clarification, transfer supernatant is in new centrifuge tube.
Staphylococcus aureuses that above-described embodiment is extracted, pseudomonas aeruginosa, actinomycetes, Oryza sativa L. Huang sporangium and
Vibrio electrophoretic effects are referring to Fig. 1 and Fig. 2.
Claims (1)
1. a kind of extracting method of the microbial gene dna rich in polysaccharide is it is characterised in that comprise the steps:
1) take appropriate thalline, add 1.5ml separate nucleic acid buffer and 20ul 2 mercapto ethanol, concussion mixes, and puts 90 DEG C of water-baths
Middle 10min, period overturns and mixes several times；
2) take out, 6000r/min centrifugation 10min in centrifuge, now it can be seen that a large amount of polysaccharide exists in solution；
3) gently suction out supernatant with pipettor, be careful not to be drawn onto polysaccharide；
4) add 1ml 1x pbs solution, 60hz on beveller, shake 2s, 10000rpm is centrifuged 2min, abandoned with soft suction of rifle
Clearly；
5) 4 are repeated) step 1 time；
6) add 1ml 1x pbs solution, shaken with handss and mix, 10000rpm is centrifuged 2min, abandon supernatant with soft suction of rifle；
7) repeat step 6) 1 time；
8) after cleaning supernatant, add 500ul stes, concussion mixes, and adds 500ul phenol chloroform, shakes 30s；
9) 12000rpm/min, is centrifuged 5min；
10) careful transfer supernatant, to new centrifuge tube, adds 5ul rnasea, and after mixing of turning upside down, room temperature places 5min；
11) 50ul magnetic bead, 500ul binding buffer are added, concussion mixes；
12) combine 5min on blood blending instrument；
13) it is placed on magnetic frame, after supernatant clarification, inhale and abandon supernatant；
14) add 1ml 80% ethanol, after standing 30s, remove ethanol；
15) after of short duration centrifugation, clean ethanol, add appropriate aquesterilisa, piping and druming mixes；
16) room temperature places 2min, is placed on magnetic frame, and after supernatant clarification, transfer supernatant is in new centrifuge tube.
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| CN201610984531.8A CN106350511A (en) | 2016-11-09 | 2016-11-09 | Extraction method for polysaccharides-enriched microbial genome DNA |
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Citations (4)
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| CN101824450A (en) * | 2010-04-23 | 2010-09-08 | 北京博迈世纪生物技术有限公司 | Kit for extracting bacterial genome based on magnetic bead and extraction method thereof |
| CN104450684A (en) * | 2014-12-26 | 2015-03-25 | 南京中科神光科技有限公司 | Kit for extracting nucleic acid from bacteria by using paramagnetic particle method and extracting method |
| CN105132410A (en) * | 2015-09-24 | 2015-12-09 | 上海派森诺生物科技有限公司 | Extraction method of microorganism genome DNA |
| CN105368815A (en) * | 2015-11-25 | 2016-03-02 | 上海派森诺生物科技股份有限公司 | Extracting method of polysaccharide and polyphenol plant genomes |
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2016
- 2016-11-09 CN CN201610984531.8A patent/CN106350511A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101824450A (en) * | 2010-04-23 | 2010-09-08 | 北京博迈世纪生物技术有限公司 | Kit for extracting bacterial genome based on magnetic bead and extraction method thereof |
| CN104450684A (en) * | 2014-12-26 | 2015-03-25 | 南京中科神光科技有限公司 | Kit for extracting nucleic acid from bacteria by using paramagnetic particle method and extracting method |
| CN105132410A (en) * | 2015-09-24 | 2015-12-09 | 上海派森诺生物科技有限公司 | Extraction method of microorganism genome DNA |
| CN105368815A (en) * | 2015-11-25 | 2016-03-02 | 上海派森诺生物科技股份有限公司 | Extracting method of polysaccharide and polyphenol plant genomes |
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| 丁晓东等: "从顽拗植物荔枝中提取基因组DNA技术的研究", 《应用与环境生物学报》 * |
| 孙璐宏等: "植物基因组DNA提取与纯化研究进展", 《西北林学院学报》 * |
| 陈大明等: "一种木本果树基因组DNA提取方法研究", 《浙江农业大学学报》 * |
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