CN106480016A - A kind of method of extracting low-copy plasmid - Google Patents
A kind of method of extracting low-copy plasmid Download PDFInfo
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- CN106480016A CN106480016A CN201610864971.XA CN201610864971A CN106480016A CN 106480016 A CN106480016 A CN 106480016A CN 201610864971 A CN201610864971 A CN 201610864971A CN 106480016 A CN106480016 A CN 106480016A
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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
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Abstract
The invention discloses a kind of method of extracting low-copy plasmid.A kind of method of extracting low-copy plasmid, it is characterized in that picking single bacterium colony is inoculated in the rich medium test tube containing corresponding antibiotic, in 37 DEG C, 220rpm, cultivate 14 17h to growing logarithmic (log) phase, the bacterium solution of culture is extended to the rich medium that 300ml contains corresponding antibiotic, 37 DEG C, 220rpm, cultivating 2.5 3h to OD600 is 1.5 1.7, add the chloromycetin of final concentration of 150～200 μ g/ml, in 37 DEG C, 220rpm inducing culture about 14 20h, then conventional method for extracting is stripped.The described preferred alkaline lysises of conventional method for extracting.The present invention is induced by chloromycetin, substantially increases the extracting amount of low-copy plasmid, successfully reduces genome pollution probability of happening, improving product First Pass Yield.
Description
Technical field
The invention belongs to biology field, it is related to a kind of method of extracting low-copy plasmid.
Background technology
Present plasmid extraction usually utilizes alkaline lysises, is the degeneration based on chromosomal DNA and plasmid DNA and renaturation
Difference reach and separate purpose, high pH makes plasmid DNA and chromosomal DNA degeneration, precipitating proteins simultaneously, then pH value is adjusted to
Property, plasmid DNA is less, and it is easy to renaturation becomes double-strand, and chromosomal DNA is larger, tangles and reticulates insoluble matters, such that it is able to logical
Cross and be centrifuged off.In the case that plasmid copy number is relatively low, extract plasmid with conventional alkaline lysises, yield is relatively low, be not suitable for big
Amount extracting, has chloromycetin can increase the document of extracting yield, but universal less, the inapplicable a large amount of plasmid extraction of scale.
Content of the invention
The purpose of the present invention is the above-mentioned deficiency for prior art, provides a kind of side of a large amount of extracting low-copy plasmid
Method.
The purpose of the present invention can be achieved through the following technical solutions：
A kind of method of extracting low-copy plasmid contains corresponding antibiosis it is characterised in that picking single bacterium colony is inoculated in 3 6ml
In the rich medium test tube of element, in 37 DEG C, 220rpm, cultivate 14 17h to growing logarithmic (log) phase, the bacterium solution of culture is extended to
300ml contains the rich medium of corresponding antibiotic, 37 DEG C, 220rpm, and culture 2.5 3h to OD600 are 1.5 1.7, add eventually
Concentration is the chloromycetin of 150～200 μ g/ml, and in 37 DEG C, 220rpm inducing culture about 14 20h, then conventional method for extracting enters
Row extracting.The described preferred alkaline lysises of conventional method for extracting.
Beneficial effect：
The present invention is induced by chloromycetin, substantially increases the extracting amount of low-copy plasmid, successfully reduces genome pollution
Probability of happening, improving product First Pass Yield.
Specific embodiment
Embodiment 1
Taking extract the plasmid of 300 μ g pET series as a example：
1 chooses speckle shakes bacterium：
17：30 on the LB flat board having converted the preferable single bacterium colony of picking growing way (circular, full, non-solid speckle) in
4ml contains the rich medium (formula of corresponding antibiotic：Abundant 1L sodium chloride 0.5g potassium dihydrogen phosphate 3g ammonium sulfate 5g 12 water
With disodium hydrogen phosphate 15g soy peptone 10g yeast powder 20g), in 37 DEG C, 220rpm, incubated overnight is to next day 8：30(OD600
≈4).
2 amplification culture
The 4ml bacterium solution of incubated overnight is extended to the rich medium that 300ml contains corresponding antibiotic, 37 DEG C, 220rpm,
Culture about 3h, to OD600 ≈ 1.5-1.7.
3 inductions
Add the chloromycetin of final concentration of 170 μ g/ml, in 37 DEG C, 220rpm inducing culture about 14-20h.
4 extractings
1) 500mL Centrifuge Cup receives bacterium；
2) add 10ml solution P1 suspension thalline, be vortexed concussion 30s, plus 200 μ l RNase (10mg/ml) are in the bacterium suspending
Body；
3) 10ml solution P2 cracking thalline, mixing of turning upside down are added, operation must be soft；
4) plus 10ml solution S 3 turns upside down mixings, standing 10min；
5) 7500rpm centrifugation 15min；
6) supernatant is filtered with filter paper into vial；
7) add 1/3V supernatant (10ml) isopropanol in vial, fully mix；
8) supernatant after filtering is added adsorption column SD5004,7500rpm, 1min；
9) 7.2ml Wash Buffer washes post twice；
10) abandon waste liquid, adsorption column is placed back in collecting pipe, dally 2min；
11) adsorption column is placed in a new 50ml centrifuge tube, volatilize in superclean bench ethanol 10min；
12) 1mL TE (EF) Buffer (65 DEG C of preheatings) is added in the central authorities of adsorption column, stands 1min；
13) 7500rpm centrifugation 2min to 50mL sterile centrifugation tube；
14) plasmid DNA is transferred to 2mL sterile centrifugation tube；
15) repeat step 4.12-4.14 is once；
16) remove endotoxin.
Explanation：
Using chloromycetin induction, finally give 300ug plasmid
Directly it is stripped using normal method, finally had to 100ug plasmid
Normal method extraction steps：
Taking extract the plasmid of 300 μ g pET series as a example：
1 chooses speckle shakes bacterium：
16：The 30 preferable single bacterium colony of picking growing way (circular, full, non-solid speckle) expansions on the LB flat board having converted
Contain the rich medium of corresponding antibiotic, 37 DEG C, 220rpm to 300ml, cultivate about 14h.
2 extractings
1) 500mL Centrifuge Cup receives bacterium；
2) add 10ml solution P1 suspension thalline, be vortexed concussion 30s, plus 200 μ l RNase (10mg/ml) are in the bacterium suspending
Body；
3) 10ml solution P2 cracking thalline, mixing of turning upside down are added, operation must be soft；
4) plus 10ml solution S 3 turns upside down mixings, standing 10min；
5) 7500rpm centrifugation 15min；
6) supernatant is filtered with filter paper into vial；
7) add 1/3V supernatant (10ml) isopropanol in vial, fully mix；
8) supernatant after filtering is added adsorption column SD5004,7500rpm, 1min；
9) 7.2ml Wash Buffer washes post twice；
10) abandon waste liquid, adsorption column is placed back in collecting pipe, dally 2min；
11) adsorption column is placed in a new 50ml centrifuge tube, volatilize in superclean bench ethanol 10min；
12) 1mL TE (EF) Buffer (65 DEG C of preheatings) is added in the central authorities of adsorption column, stands 1min；
13) 7500rpm centrifugation 2min to 50mL sterile centrifugation tube；
14) plasmid DNA is transferred to 2mL sterile centrifugation tube；
15) repeat step 2.12-2.14 is once；
16) remove endotoxin.
It is stripped with the inventive method, for the plasmid of pET, the ratio of genome pollution reduces about
90%.
Claims (2)
1. a kind of method of extracting low-copy plasmid contains corresponding antibiotic it is characterised in that picking single bacterium colony is inoculated in 3 6ml
Rich medium test tube in, in 37 DEG C, 220rpm, cultivate 14 17h to growing logarithmic (log) phase, the bacterium solution of culture be extended to
300ml contains the rich medium of corresponding antibiotic, 37 DEG C, 220rpm, and culture 2.5 3h to OD600 are 1.5 1.7, add eventually
Concentration is the chloromycetin of 150～200 μ g/ml, and in 37 DEG C, 220rpm inducing culture about 14 20h, then conventional method for extracting enters
Row extracting.
2. method according to claim 1 is it is characterised in that described conventional method for extracting is alkaline lysises.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610864971.XA CN106480016A (en) | 2016-09-29 | 2016-09-29 | A kind of method of extracting low-copy plasmid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610864971.XA CN106480016A (en) | 2016-09-29 | 2016-09-29 | A kind of method of extracting low-copy plasmid |
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| Publication Number | Publication Date |
|---|---|
| CN106480016A true CN106480016A (en) | 2017-03-08 |
Family
ID=58268165
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| Application Number | Title | Priority Date | Filing Date |
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| CN201610864971.XA Withdrawn CN106480016A (en) | 2016-09-29 | 2016-09-29 | A kind of method of extracting low-copy plasmid |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999053044A2 (en) * | 1998-04-10 | 1999-10-21 | Genset | High throughput dna sequencing vector |
| JP2007037498A (en) * | 2005-08-04 | 2007-02-15 | Tatsuhiko Kobayashi | Method for producing protein |
| CN102559729A (en) * | 2012-03-08 | 2012-07-11 | 中山大学 | Expression plasmid for integrating and evolving chromosome |
-
2016
- 2016-09-29 CN CN201610864971.XA patent/CN106480016A/en not_active Withdrawn
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999053044A2 (en) * | 1998-04-10 | 1999-10-21 | Genset | High throughput dna sequencing vector |
| JP2007037498A (en) * | 2005-08-04 | 2007-02-15 | Tatsuhiko Kobayashi | Method for producing protein |
| CN102559729A (en) * | 2012-03-08 | 2012-07-11 | 中山大学 | Expression plasmid for integrating and evolving chromosome |
Non-Patent Citations (2)
| Title |
|---|
| 徐小元等: "一种改进简便的质粒DNA提取方法", 《兰州医学院学报》 * |
| 王友如: "大肠杆菌质粒DNA提取方法的优化", 《生物技术》 * |
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| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20180126 Address after: 212132 Zhenjiang city Jiangsu province area Dingmao after fifteen Road No. 99 Building No. 30 Applicant after: Jiangsu Genscript Biotechnology Co. Ltd. Address before: Jiangning District of Nanjing City, Jiangsu province 211100 Science Park Yong Xilu No. 28 (Zijin Mountain) Applicant before: Nanjing Genscript Biotechnology Co., Ltd. |
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| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information | ||
| CB02 | Change of applicant information |
Address after: 212132 54 Building, 99 Dingmaojing 15th Road, Zhenjiang New District, Jiangsu Province Applicant after: Jiangsu Genscript Biotechnology Co. Ltd. Address before: 212132 Building No. 30, 99 Dingmaojing 15th Road, Zhenjiang New District, Jiangsu Province Applicant before: Jiangsu Genscript Biotechnology Co. Ltd. |
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| WW01 | Invention patent application withdrawn after publication | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20170308 |