CN1958800B - Device for extracting plasmid DNA - Google Patents
Device for extracting plasmid DNA Download PDFInfo
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- CN1958800B CN1958800B CN200610123715A CN200610123715A CN1958800B CN 1958800 B CN1958800 B CN 1958800B CN 200610123715 A CN200610123715 A CN 200610123715A CN 200610123715 A CN200610123715 A CN 200610123715A CN 1958800 B CN1958800 B CN 1958800B
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Abstract
This invention discloses method and apparatus for extracting plasmid DNA. The method comprises: culturing engineering bacteria, decomposing engineering bacteria, extracting and purifying plasmid. The method utilizes improved TB culture medium to culture engineering bacteria, thus can improve the quality of engineering bacteria and plasmid. The method utilizes inexpensive cheese cloths and cellulose ester millpore filtration membrane for separation, thus can save the cost and time, and can obtain high-purity product with high yield. The method utilizes 50-60 deg.C double distilled water or TE to dissolve the plasmid, thus can increase the yield and efficiency. The method can extract plasmid in different scales, and the extraction process is simple. The plasmid has high quality and yield, and can be used in digestion, ligation, transformation, and production of nucleic acid vaccines.
Description
Technical field
The invention belongs to field of molecular biotechnology, particularly a kind of extracting method of plasmid DNA and extraction element thereof.
Background technology
The extraction of plasmid DNA and purifying are one of the most basic molecular biological steps, the plasmid extracting method of the classics in the molecular cloning relates to a lot centrifugal, limited the scale of extracting plasmid, from the 500mL culture, can only gather in the crops 2～5mg plasmid, and the purge process complexity, and used poisonous and hazardous material.Along with the fast development of dna vaccination and gene recombination drug research and application, in continuous increase, conventional prepared in laboratory and method thereof have been difficult to satisfy to the demand of recombinant plasmid.Also having some extensive methods of extracting at present is to use various types of affinity columns, and extraction effect is better, but expense is very high, also is difficult for the expansion scale.
Summary of the invention
Weak point at present existing plasmid extracting technology existence; primary and foremost purpose of the present invention has been to provide a kind of extracting method of plasmid DNA; this method has been simplified the process that plasmid extracts, and has solved the problem that plasmid is extracted in mass-producing, and output height, purity are good.
Another object of the present invention is to provide the extraction element of above-mentioned plasmid DNA.
Purpose of the present invention is achieved through the following technical solutions: a kind of extracting method of plasmid DNA comprises the steps:
(1) cultivation of engineering bacteria: engineering bacteria is inoculated in improvement TB (Terrific Broth) liquid nutrient medium, about 12～14 hours of 37 ℃, 300 rev/mins shaking culture, the culture that obtains logarithm late period is that the OD value is about 0.6 culture; The inoculation volume percent is 0.5～1%, and described improvement TB liquid nutrient medium is: with component protein peptone 12g, yeast extract 24g, glycerine 4mL is dissolved in the 900mL water, autoclaving after each components dissolved is cooled to 60 ℃, adds the 170mmol/LKH of 100mL through sterilization again
2PO
4With 720mmol/L Na
2HPO
4Solution.
(2) cracking of engineering bacteria: the culture of getting in the step (1) adopts alkaline lysis cracking engineering bacteria.
(3) the extraction purifying of plasmid: split product filters by 4～6 layers of cheese cloth, collect filtrate, add the RNA enzyme, making final concentration is 20ug/mL, 37 ℃ act on 20 minutes, the Virahol that in filtrate, adds 0.6 filtrate volume then, room temperature (20～25 ℃) was placed 2 minutes, and the precipitation plasmid is that 0.22 μ m mixed cellulose ester microporous membrane filters with the aperture then, discard filtrate, plasmid is stayed on the film, the alcohol flushing mixed cellulose ester microporous membrane with 70% 2～3 times, air-dry filter membrane, use 50～60 ℃ distilled water or the TE of pH8.0 (Tris/EDTA) dissolving plasmid DNA at last, can obtain plasmid DNA.
Described engineering bacteria comprises DH5 or TOP10 etc.
In order to realize the present invention better, the 170mmol/L KH of described sterilization
2PO
4And 720mmol/LNa
2HPO
4Solution be prepared from as follows: with 2.31g KH
2PO
4With 12.54g Na
2HPO
4Be dissolved in the water of 100ml autoclaving or with the membrane filtration degerming of 0.22 μ m.
The present invention also provides the extraction element of above-mentioned plasmid DNA, this device comprises that alkaline lysis precipitates with container (4), contains plasmid container (8) and filter with container (1), plasmid, described alkaline lysis links to each other by the filter (2) that cheese cloth is housed with container (4) with the plasmid precipitation with container (1), the plasmid precipitation links to each other by the filter (5) that mixed cellulose ester microporous membrane is housed with container (8) with the Sheng plasmid with container (4), and the plasmid precipitation is equipped with valve (6) with container (4), Sheng plasmid with container (8) top; The plasmid precipitation is passed through the external vacuum pump of first pipe connecting (3) with container (4), and first pipe connecting (3) is equipped with sealed valve; Contains plasmid and pass through the external vacuum pump of second pipe connecting (7), and second pipe connecting (7) is equipped with sealed valve with container (8).
In order to realize the present invention better, described alkaline lysis all adopts stainless steel to make with container (4) and Sheng plasmid with container (8) with container (1), plasmid precipitation.
This device can be designed to different size specifications as required, and the plasmid precipitation is used 2～6 times of container (1) volume for alkaline lysis with container (4).The described filter (5) that the filter (2) of cheese cloth is housed and mixed cellulose ester microporous membrane is housed all can be interchangeable membrane filter.The described filter membrane that the filter (2) of cheese cloth is housed is 4～6 layers a cheese cloth, and the described mixed cellulose ester microporous membrane aperture that the filter (5) of mixed cellulose ester microporous membrane is housed is 0.22 μ m.
The present invention improves alkaline lysis plasmid extraction method; saved the centrifugal step; simplified the process that plasmid extracts; and solved the problem of mass-producing extraction plasmid; and output height; can extract about 50mg plasmid from reaching every liter of nutrient solution, purity is good, and the mean value of OD260/280 is 1.89.
The present invention compared with prior art has following advantage and beneficial effect:
The present invention has adopted TB (Terrific Broth) the culture medium culturing engineering bacteria of improvement, has improved the output of bacterium and plasmid, apparently higher than the engineering bacteria with the fermentation of LB substratum; The present invention has utilized low price, cheese cloth commonly used and the mixed cellulose ester microporous membrane of 0.22 μ m to replace the filtering step in the classical way, cost and time have been saved, and output and purity can reach the requirement of general molecular biology experiment than higher; The present invention has utilized 50～60 ℃ TE or distilled water dissolving plasmid, has improved output and efficient; The plasmid that the present invention can be used for various scales extracts, from several milliliters to several liters, even more massive plasmid extracts; The plasmid that the present invention can satisfy different scales extracts requirement, and leaching process is simple, and yield plasmid and quality are all fine, can be used for enzyme and cut, connect and transform, and can also satisfy the production of nucleic acid vaccine.
Description of drawings
Fig. 1 is the influence figure of improvement Terrific Broth to engineering bacteria output.
The plasmid agarose electrophoresis collection of illustrative plates that Fig. 2 extracts for the present invention.
Fig. 3 cuts evaluation figure for the enzyme of the recombinant plasmid that the present invention extracts.
Fig. 4 is the structural representation of extraction element of the present invention.
Embodiment
The present invention is described in further detail below in conjunction with drawings and Examples, but embodiments of the present invention are not limited thereto.
Embodiment 1: a small amount of preparation of plasmid, from 3mL engineering bacteria DH5 culture, extract plasmid pMD-18T, and can extract plasmid 296 μ g, the value of OD260/280 is between 1.8～2.0.
(1) cultivation of engineering bacteria: single bacterium colony of growing on the picking LB solid medium (engineering bacteria DH5), be inoculated in 2.0ml improvement TB (the containing 60 μ g/mL penbritins) liquid nutrient medium, 37 ℃, 300 rev/mins thermal agitation overnight incubation (about 12～14h), obtain the logarithm culture in late period (the OD value is about 0.6), the inoculation volume percent is 0.5～1%.Improvement TB liquid nutrient medium: with component protein peptone 12g, yeast extract 24g, glycerine 4mL are dissolved in the 900mL water, and autoclaving after each components dissolved is cooled to 60 ℃, add the 170mmol/L KH of 100mL through sterilization again
2PO
4With 720mmol/L Na
2HPO
4Solution (be about to 2.31g KH
2PO
4With 12.54g Na
2HPO
4Be dissolved in the water of 100ml, autoclaving or with the membrane filtration degerming of 0.22 μ m).Add nutrient solution (improvement TB liquid nutrient medium) and be no more than 1/4th of used culture vessel.
(2) cracking of engineering bacteria: the alkaline lysis cracking engineering bacteria that adopts " molecular cloning ".
Get the middle 3ml culture of step (1) and go in the micro-centrifuge tube, the centrifugal 8000g * 1min of room temperature abandons supernatant, and centrifuge tube is inverted, and liquid is flow to end as far as possible.Bacterium (engineering bacteria of turning out) precipitation is resuspended in the solution I of 100 μ l precoolings, and thermal agitation makes thalline disperse mixing.Add the freshly prepared solution II of 200 μ l, put upside down mixing (not thermal agitation) for several times, and centrifuge tube is positioned over 2～3min on ice, make cytolemma cracking (solution II is a lysate, so bacterium liquid becomes clearly gradually in the centrifuge tube).The solution III that adds 150 μ l precoolings will be managed gentleness and be put upside down mixing for several times, see white flocks, can place 3～5min on ice.Solution III is a neutralization solution, this moment the plasmid DNA renaturation, karyomit(e) and protein irreversible denaturation form soluble mixture, simultaneously K
+Make SDS-albumen composition precipitation.
Mentioned reagent is prepared as follows:
Solution I: 50mM glucose, 25mM Tris-HCl (pH 8.0), 10mM EDTA (pH 8.0), 1M Tris-HCl (pH 8.0) 12.5ml, 0.5M EDTA (pH 8.0) 10ml, glucose 4.730g adds ddH
2O to 500ml is at 10lbf/in
2(6.895 * 10
4) autoclaving 15min, be stored in 4 ℃.
Solution II: 0.2NNaOH, 1%SDS, 2N NaOH 1ml, 10%SDS 1ml adds ddH
2O to 10ml.Interim preparation before using.
Solution III: Potassium ethanoate (KAc) damping fluid, pH 4.8,5M KAc 300ml, Glacial acetic acid 57.5ml adds ddH
2O to 500ml.4 ℃ of preservations are standby.
(3) the extraction purifying of plasmid: split product filters by 4～6 layers of cheese cloth, utilize syringe filter and syringe to finish, collect filtrate, add the RNA enzyme, final concentration is 20ug/mL, 37 ℃ act on 20 minutes, add the Virahol of 0.6 filtrate volume then in filtrate, room temperature was placed 2 minutes, in the inhalation syringe, mixed cellulose ester microporous membrane with aperture 0.22 μ m filters then, discard filtrate, plasmid is stayed on the CN-CA, the alcohol flushing mixed cellulose ester microporous membrane with 70% 2～3 times, abundant air-dry filter membrane is used 50～60 ℃ distilled water (or TE of 50～60 ℃ pH8.0) dissolving plasmid DNA at last as required.The gained plasmid carries out enzyme and cuts experiment such as connection conversion (see figure 3).
As shown in Figure 1, be the influence of improvement Terrific Broth to engineering bacteria output.A1 is the engineering bacteria that 1.5mL improvement Terrific Broth (improvement TB liquid nutrient medium) turns out, and A2 is the engineering bacteria that LB turns out under the same terms, its OD value after measured, and the bacteria content of A1 is 5 times of A2.
As shown in Figure 2, the plasmid agarose electrophoresis collection of illustrative plates that extracts for the present invention.The plasmid that extracts is pAdTrack, and intermediary is the marker of 2000bp, and no RNA pollutes.
As shown in Figure 3, the enzyme of the recombinant plasmid that extracts for the present invention is cut evaluation figure.Cut the requirement that the plasmid that confirms present method extraction can satisfy general molecular biology experiment by enzyme.This enzyme that is the plasmid pAdTrack that contains pig circular ring virus 2 virus gene ORF2, ORF1+2 and ORF1 that extracts carries out is cut, and has obtained corresponding fragment, proves that plasmid that this method is extracted can satisfy the basic demand of molecular biosciences field test.
Embodiment 2: the mass preparation of plasmid
With the method culturing engineering bacterium among the embodiment 1, nutrient solution that requirement adds is no more than 1/4th, 300 rev/mins of concuss of used culture vessel and cultivated 12 hours.Utilize the device of extraction plasmid of the present invention to be prepared.Can extract the 50mg plasmid from the 500mL engineering bacteria, the value of OD260/280 is between 1.8～1.9.Wherein add solution I 60mL, solution II 120mL, solution III 90mL.
Embodiment 3
Select as required, this device can be designed to different size specifications.As shown in Figure 4, the extraction element of plasmid DNA of the present invention, this device comprises that alkaline lysis precipitates with container 4, contains plasmid container 8 and filter with container 1, plasmid, described alkaline lysis links to each other by the filter 2 that cheese cloth is housed with container 4 with the plasmid precipitation with container 1, the plasmid precipitation links to each other by the filter 5 that mixed cellulose ester microporous membrane is housed with container 8 with the Sheng plasmid with container 4, and the plasmid precipitation is equipped with valve 6 with container 4, Sheng plasmid with container 8 tops; The plasmid precipitation is passed through pipe connecting 3 external vacuum pumps with container 4, and sealed valve 6 is housed; Contain plasmid and pass through pipe connecting 7 external vacuum pumps, and sealed valve 6 is housed with container 8; Described alkaline lysis adopts stainless steels to make with container 4 and Sheng plasmid with container 8 with container 1, plasmid precipitation.This device can be designed to different size specifications as required, and the plasmid precipitation is 2～6 times with container 1 volume of alkaline lysis with container 4, and the filter 2 that cheese cloth is housed all can be interchangeable membrane filter with the filter 5 that mixed cellulose ester microporous membrane is housed.The described filter membrane that the filter 2 of cheese cloth is housed is 4～6 layers a cheese cloth, and the described mixed cellulose ester microporous membrane aperture that the filter 5 of mixed cellulose ester microporous membrane is housed is 0.22 μ m.
Alkaline lysis links to each other with the filter 2 that cheese cloth is housed with container 1, the junction sealing, lysate is precipitating under the effect of the vacuum pump that links to each other with container 4 with plasmid, filter 2 through cheese cloth is housed enters plasmid precipitation container 4, entering plasmid precipitates with the valve 6 of closing its top behind the container 4, in precipitating with the filtrate in the container 4, plasmid adds RNA enzyme and Virahol by pipe connecting 3, with the effect of containing the vacuum pump that plasmid is connected with container 8 under, enter Sheng plasmid container 8 through the filter 5 that aperture 0.22 μ m mixed cellulose ester microporous membrane is housed, the plasmid precipitation uses container 8 respectively by pipe connecting 3 with container 4 and Sheng plasmid, pipe connecting 7 external vacuum pumps, and sealed valve is housed.
Technical process in the extraction element of plasmid DNA of the present invention is as follows: the engineering bacteria of collecting is put into alkaline lysis container 1, successively add solution I according to the aforementioned bases cracking process, solution II, solution III, alkaline lysis enters plasmid precipitation container 4 by the filter 2 (filter membrane is 4～6 layers a cheese cloth) that cheese cloth is housed with the lysate in the container 1 under the effect of vacuum pump, add Virahol by pipe connecting 3, the plasmid post precipitation, with contain the vacuum pump effect that plasmid links to each other with container 8 under, by the filter 5 of 0.22 μ m mixed cellulose ester microporous membrane is housed, plasmid is stayed on the film, again to the ethanol of plasmid precipitation with interpolation 70% in the container 4, twice of flushing plasmid, change a Sheng plasmid container 8 that cleaning is aseptic, dissolve plasmid in the plasmid precipitation with adding distilled water in the container 4, by the vacuum pump effect, plasmid is collected the aseptic Sheng plasmid of cleaning container 8.
As mentioned above, can realize the present invention preferably.
Claims (6)
1. the extraction element of a plasmid DNA, this device comprises that alkaline lysis precipitates with container (4), contains plasmid container (8) and filter with container (1), plasmid, it is characterized in that, described alkaline lysis links to each other by the filter (2) that cheese cloth is housed with container (4) with the plasmid precipitation with container (1), the plasmid precipitation links to each other by the filter (5) that mixed cellulose ester microporous membrane is housed with container (8) with the Sheng plasmid with container (4), and the plasmid precipitation is equipped with valve (6) with container (4), Sheng plasmid with container (8) top; The plasmid precipitation is passed through the external vacuum pump of first pipe connecting (3) with container (4), and first pipe connecting (3) is equipped with sealed valve; Contain plasmid and pass through the external vacuum pump of second pipe connecting (7), and second pipe connecting (7) is equipped with sealed valve with container (8).
2. the extraction element of a kind of plasmid DNA according to claim 1 is characterized in that, described alkaline lysis all adopts stainless steel to make with container (4) and Sheng plasmid with container (8) with container (1), plasmid precipitation.
3. the extraction element of a kind of plasmid DNA according to claim 1 is characterized in that, the plasmid precipitation is used 2～6 times of container (1) volume for alkaline lysis with container (4).
4. the extraction element of a kind of plasmid DNA according to claim 1 is characterized in that, the described filter (5) that the filter (2) of cheese cloth is housed and mixed cellulose ester microporous membrane is housed is interchangeable membrane filter.
5. the extraction element of a kind of plasmid DNA according to claim 1 is characterized in that, the described filter membrane that the filter (2) of cheese cloth is housed is 4～6 layers a cheese cloth.
6. the extraction element of a kind of plasmid DNA according to claim 1 is characterized in that, the described mixed cellulose ester microporous membrane aperture that the filter (5) of mixed cellulose ester microporous membrane is housed is 0.22 μ m.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200610123715A CN1958800B (en) | 2006-11-23 | 2006-11-23 | Device for extracting plasmid DNA |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200610123715A CN1958800B (en) | 2006-11-23 | 2006-11-23 | Device for extracting plasmid DNA |
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| Publication Number | Publication Date |
|---|---|
| CN1958800A CN1958800A (en) | 2007-05-09 |
| CN1958800B true CN1958800B (en) | 2010-05-12 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200610123715A Expired - Fee Related CN1958800B (en) | 2006-11-23 | 2006-11-23 | Device for extracting plasmid DNA |
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| Country | Link |
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| CN (1) | CN1958800B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115885030A (en) * | 2020-08-17 | 2023-03-31 | 南京金斯瑞生物科技有限公司 | Automated integration system for continuous commercial scale plasmid production |
| CN115404154A (en) * | 2021-05-10 | 2022-11-29 | 艾棣维欣(苏州)生物制品有限公司 | Device for extracting plasmid DNA from bacteria |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1187196A (en) * | 1995-06-08 | 1998-07-08 | 普罗金工业有限公司 | Method and apparatus for DNA extraction |
| DE20111082U1 (en) * | 2001-07-06 | 2001-10-31 | Macherey Nagel Gmbh & Co Hg | Device for the treatment of biomolecules |
-
2006
- 2006-11-23 CN CN200610123715A patent/CN1958800B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1187196A (en) * | 1995-06-08 | 1998-07-08 | 普罗金工业有限公司 | Method and apparatus for DNA extraction |
| DE20111082U1 (en) * | 2001-07-06 | 2001-10-31 | Macherey Nagel Gmbh & Co Hg | Device for the treatment of biomolecules |
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| Publication number | Publication date |
|---|---|
| CN1958800A (en) | 2007-05-09 |
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