CN1654660A - Kit for quick extraction and purification of plasmid and its application - Google Patents
Kit for quick extraction and purification of plasmid and its application Download PDFInfo
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- CN1654660A CN1654660A CN 200410047132 CN200410047132A CN1654660A CN 1654660 A CN1654660 A CN 1654660A CN 200410047132 CN200410047132 CN 200410047132 CN 200410047132 A CN200410047132 A CN 200410047132A CN 1654660 A CN1654660 A CN 1654660A
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Abstract
The present invention discloses one kind of fast plasmid extracting and purifying reagent kit and bacterium plasmid extracting and purifying process. The reagent kit includes the following separately maintained liquid: no-alkali enzyme-containing bacterium cracking liquid, DNA binding column pre-treating liquid, DNA plasmid binding liquid, cleaning liquid and eluting liquid. The bacterium plasmid extracting and purifying process with the kit includes the following steps: cracking bacterium, pre-treating DNA binding column, binding plasmid DNA, cleaning and eluting. The present invention makes it possible to complete the bacterium plasmid extracting and purifying process in 3-5 min and to complete mass sample extraction, and the obtained plasmid DNA has A260/A280 up to 1.7-2.0.
Description
Technical field
The present invention relates to a kind of rapid plasmid extracting and purifying test kit and application thereof, especially relate to a kind of bacterial plasmid extracting and purifying test kit and application thereof.
Background technology
The plasmid of bacterium is the important carrier and the instrument of DNA reorganization, and the separation and purification plasmid is the requisite means of Modern Molecular Biotechnology.The separation of existing plasmid mainly contains two methods: the one, by the alkaline lysis of Birnbiom and Doly creation, the 2nd, by the boiling method of Holmes and Quigley invention, also have some to develop on this basis and other method of coming as PEG method, cesium chloride density gradient centrifugation etc.At present the plasmid extraction method of generally using is to serve as that the basis is in conjunction with the affine character of silicon-dioxide DNA with the alkaline lysis method, add scavenging solution obtains purifying with the method for centrifugal or vacuum extraction plasmid, this method has creatively been improved the extracting and purifying process of plasmid, and it has been widely used in current medical science, agronomy and other molecular biology research field.Institute's popular various " miniprep ", " midiprep ", " maxiprep " are the various plasmid purification test kits that businessman provided in the market, the most classical with Qiagen company wherein, its test kit comprises seven kinds of self-existent different liqs reagent, carry out plasmid purification with it, specifically may further comprise the steps: collect thalline, resuspended, alkaline lysis, neutralisation, the long-time centrifugal removal of impurity, DNA in conjunction with, clean, wash-out etc.This method has shortened the time of extracting and purifying plasmid, and the method method relatively in the past that makes plasmid purification is simply and effective, and the general extracting and purifying time of spending is about 30 minutes, therefore for the scientific research personnel provides more convenience, has saved the time.Yet this method need be carried out resuspended, the neutralisation of thalline and numerous and diverse step such as centrifugal for a long time, and the time of cost is still longer, and institute's cost of spending is also higher, and the extracting of not too suitable a large amount of samples has also limited the process of its automatization.CN1546516A discloses a kind of plasmid DNA and has extracted and purification process, and extraction and purifying time are also very long, and efficient is low.
Summary of the invention
The objective of the invention is to overcome the defective that existing plasmid extraction purification process exists, provide a kind of formation simple, extracting and purifying is easy and simple to handle, plasmid extraction purification kit that efficient is higher and plasmid extraction purification process.
The objective of the invention is to realize by the following technical solutions:
Test kit of the present invention comprises following four kinds of self-existent liquid at least: cellular lysate liquid, DNA column pretreatment liquid, DNA plasmid is in conjunction with liquid, scavenging solution; Preferred version is also to dispose elutriant.
Described cellular lysate liquid formula: glucose 10mM-1M, Trisaminomethane hydrochloric acid 20mM-1M, ethylenediamine tetraacetic acid (EDTA) 10mM-500mM, sodium bicarbonate 100mM-500mM, Triton 1%-50% (volume/volume), sodium laurylsulfonate 1%-25% (weight/volume), N,O-Diacetylmuramidase 2mg/ml-1g/ml, rnase 5-50mg/ml.
Described DNA column pretreatment liquid prescription: lithium chloride or sodium-chlor 10mM-5M, potassium hydroxide or sodium hydroxide 100mM-2M, sodium laurylsulfonate 5%-20% (weight/volume), hydrochloric acid 0.01M-2.5M.
Described DNA plasmid is in conjunction with the prescription of liquid: Guanidinium hydrochloride 1M-6M, sodium-chlor 10mM-100mM, morpholino ethane sulfonic acid 10mM-100mM, Virahol; Four volume proportions: 40: 40: 10: 10.
Described scavenging solution prescription: Trisaminomethane hydrochloric acid 1-50mM, ethanol 70-85% (volume).
Described elutriant is the Trisaminomethane hydrochloric acid of pH8-9 concentration 8-12mM; When not being equipped with this kind elutriant in the test kit, can replace this kind elutriant to carry out wash-out by pure water.
Each raw material is mixed by proportioning, can be prepared into described various solution.
It is as follows to use this test kit to carry out the method for bacterial plasmid extracting and purifying:
Cracking thalline: 40-100 μ l cracked solution is added 300-1000 μ l (OD600＞2) cultivate bacterium liquid, mix and place 40-80 second;
Pre-treatment DNA column: 150-300 μ l DNA column pre-treatment solution is added in the DNA column, leave heart 8-20 second, reject solution with 10000-15000;
In conjunction with plasmid DNA: 200-400 μ l DNA plasmid binding soln is added among the cracking bacterium liquid, and the centrifugal tubule of overturning is 8-12 time up and down, mixes, and among the DNA column that adding had been handled, leaves heart 10-30 second with 10000-15000;
Clean: add cleaning solution 700-900 μ l, leave heart 8-20 second, reject centrifugate, centrifugal again 50-80 second with 10000-15000.
Wash-out: add 30-40 μ l elutriant in DNA column center, leave heart 20-40 second, can obtain the high purity plasmid DNA with 10000-15000.
The plasmid DNA of using test kit of the present invention and plasmid extraction purification process to obtain detects A through using spectrophotometer
260/ A
280Value is 1.7-2.0.
Use test kit of the present invention and plasmid extraction purification process from the thalline of cultivating, to extract highly purified plasmid, can be used for following various experiment, as: enzyme is cut reorganization, order-checking, PCR or the like.
The present invention has abandoned the used alkaline lysis of plasmid extraction in the past, adopts gentle denaturing agent and enzymatic lysis method, needn't centrifugal collection thalline, and directly utilize high density (OD600＞2) to cultivate bacterium liquid (as bacillus coli DH 5 alpha, XL1-Blue etc.) and carry out cracking; Adopt brand-new solution formula pre-treatment silicon dioxide fibrous membrane, make more easy-clear of impurity such as albumen, RNA bacterium diaphragm, and the easier combination of plasmid DNA; The steps such as resuspended, neutralisation in the existing widely used classical way have also been saved; Just because of above-mentioned reason, use the present invention's test kit and plasmid extraction purification process, can in 3-5 minute, fast and effeciently finish the work of plasmid extraction purifying, shorten the extracting time greatly, save a large amount of experimental periods thereby can be the researchist; Can also be used for the batch samples extracting, in case of emergency the extracting and purifying plasmid; This method has also been established technical foundation for the automatization and extensiveization of plasmid extraction.
Embodiment
The invention will be further described below in conjunction with embodiment.
Embodiment 1
The prescription of lysate is: glucose 10mM, Trisaminomethane hydrochloric acid 20mM, ethylenediamine tetraacetic acid (EDTA) 10mM, sodium bicarbonate 100mM, Triton 1% (volume/volume), sodium laurylsulfonate 1% (weight/volume percent), N,O-Diacetylmuramidase 2mg/ml, rnase 5mg/ml.Making each sample need get 60 μ l and put into centrifugal tubule.
The prescription of DNA column pretreatment liquid is: lithium chloride 10mM, sodium hydroxide 0.001M, sodium laurylsulfonate 5%, hydrochloric acid 0.01M.Making each sample gets 250 μ l and puts into column.
The DNA plasmid in conjunction with the prescription of liquid is: Guanidinium hydrochloride 6M, and sodium-chlor 100mM, morpholino ethane sulfonic acid 100mM, Virahol, the ratio of four volumes is: 40: 40: 10: 10.Making each sample gets 350 μ l and puts into column.
The prescription of scavenging solution is: Trisaminomethane hydrochloric acid 1mM adds dehydrated alcohol 80% (volume) again.Making each sample gets 750 μ l and puts into column.
Elutriant is: Trisaminomethane hydrochloric acid 8mM, pH8.Each sample amount of getting is 40 μ l.
Use this test kit, the step of carrying out the plasmid extraction purifying is as follows:
(1) cracked solution 60 μ l is added 800 μ l (OD600＞2) bacillus coli DH 5 alpha nutrient solution, mix and placed for 80 seconds, carried out for second step simultaneously;
(2) 250 μ l pre-treatment solution are added in the DNA column, 10000 left the heart 20 seconds, reject solution;
(3) 350 μ, 1 DNA binding soln is added among the cracking bacterium liquid, the centrifugal tubule of overturning is 12 times up and down, mixes, and adds among the DNA column of having handled centrifugal 30 seconds (15000 change);
(4) add cleaning solution 750 μ l, 15000 left the heart 20 seconds, reject centrifugate, centrifugal again 80 seconds;
(5) add 40 μ l elutriants in the column center, 15000 left the heart 40 seconds, obtained the high purity plasmid DNA.
Through using spectrophotometer that extracting and purifying gained plasmid DNA is detected A
260/ A
280Value is 1.7.
Embodiment 2
The prescription of lysate is: glucose 500mM, Trisaminomethane hydrochloric acid 200mM, ethylenediamine tetraacetic acid (EDTA) 100mM, sodium bicarbonate 200mM, Triton 10% (volume/volume per-cent), sodium laurylsulfonate 10% (weight/volume percent), N,O-Diacetylmuramidase 20mg/ml, rnase 0.5mg/ml.Making each sample gets 50 μ l and puts into centrifugal tubule.
The prescription of DNA column pretreatment liquid is: sodium-chlor 2M, potassium hydroxide 0.1M, sodium laurylsulfonate 10% (weight/volume percent), hydrochloric acid 0.5M.Making each sample gets 200 μ l and puts into column.
The DNA plasmid in conjunction with the prescription of liquid is: Guanidinium hydrochloride 2.5M, sodium-chlor 3mM, morpholino ethane sulfonic acid 50mM, Virahol 100% (volume), four volume ratios: 40: 40: 10: 10.Making each sample gets 300 μ l and puts into column.
The prescription of scavenging solution is: Trisaminomethane hydrochloric acid 10mM adds dehydrated alcohol 80% (volume).Making each sample gets 600 μ l and puts into column.
Elutriant is the aminomethane hydrochloric acid of pH8.5 concentration 10mM.Each sample amount of getting is 35 μ l.
Use this test kit, the step of carrying out the plasmid extraction purifying is as follows:
(1) 50 μ l cracked solution is added 500 μ l (OD600＞2) bacillus coli DH 5 alpha nutrient solution, mix, placed for 60 seconds, carried out for second step simultaneously;
(2) 200 μ l pre-treatment solution are added in the DNA column, 12000 left the heart 10 seconds, reject solution;
(3) 300 μ l binding solns are added among the cracking bacterium liquid, the centrifugal tubule of overturning is 10 times up and down, mixes, and adds among the DNA column of having handled, and 12000 left the heart 10 seconds;
(4) add cleaning solution 600 μ l, 12000 left the heart 10 seconds, reject centrifugate, centrifugal again 60 seconds;
(5) add 35 μ l elutriants in the column center, 12000 left the heart 30 seconds, promptly obtained the high purity plasmid DNA.
Through using spectrophotometer that extracting and purifying gained plasmid DNA is detected A
260/ A
280Value is 2.0.
Embodiment 3
The prescription of lysate is: glucose 1M, Trisaminomethane hydrochloric acid 1M, ethylenediamine tetraacetic acid (EDTA) 500mM, sodium bicarbonate 500mM, Triton 50% (volume/volume), sodium laurylsulfonate 25% (weight/volume), N,O-Diacetylmuramidase 1mg/ml, rnase 50mg/ml.Making each sample gets 40 μ l and puts into centrifugal tubule.
The prescription of DNA column pretreatment liquid is: lithium chloride 5M, potassium hydroxide 2M, sodium laurylsulfonate 20% (weight/volume), hydrochloric acid 2.5M.Making each sample gets 150 μ l and puts into column.
The DNA plasmid in conjunction with the prescription of liquid is: Guanidinium hydrochloride 1M, and sodium-chlor 10mM, morpholino ethane sulfonic acid 10mM, Virahol, four volume ratios are: 40: 40: 10: 10.Making each sample gets 250 μ l and puts into column.
The recipe ratio of scavenging solution is: Trisaminomethane hydrochloric acid 50mM adds ethanol 85% (volume) again.Making each sample gets 500 μ l and puts into column.
Elutriant is a pure water.Each sample amount of getting is 30 μ l.
Use this test kit, the step of carrying out the plasmid extraction purifying is as follows:
(1) 40 μ l cracked solution is added 300 μ l (OD600＞2) XL1-Blue nutrient solution, mix and placed for 40 seconds, carried out for second step simultaneously.
(2) 150 μ l pre-treatment solution are added in the DNA column, 10000 left the heart 8 seconds, reject solution.
(3) 250 μ l binding solns are added among the cracking bacterium liquid, the centrifugal tubule of overturning is 8 times up and down, mixes, and adds among the DNA column of having handled centrifugal 10 seconds (10000 change).
(4) add cleaning solution 500 μ l, 10000 left the heart 8 seconds, reject centrifugate, centrifugal again 50 seconds.
(5) add 30 μ l pure water in the column center, 10000 left the heart 20 seconds, promptly obtained the high purity plasmid DNA.
Through using spectrophotometer that extracting and purifying gained plasmid DNA is detected A
260/ A
280Value is 1.8.
Employed DNA column is the Miniprep DNA column that Changsha An Biao Bioisystech Co., Ltd produces in the foregoing description.
Claims (4)
1, a kind of plasmid extraction purification kit is characterized in that, comprises following four kinds of self-existent liquid at least: cellular lysate liquid, DNA column pretreatment liquid, DNA plasmid is in conjunction with liquid, scavenging solution;
Described cellular lysate liquid formula: glucose 10mM-1M, Trisaminomethane hydrochloric acid 20mM-1M, ethylenediamine tetraacetic acid (EDTA) 10mM-500mM, sodium bicarbonate 100mM-500mM, Triton 1%-50% (volume/volume), sodium laurylsulfonate 1%-25% (weight/volume), N,O-Diacetylmuramidase 2mg/ml-1000mg/ml, rnase 5-50mg/ml;
Described DNA column pretreatment liquid prescription: lithium chloride or sodium-chlor 10mM-5M, potassium hydroxide or sodium hydroxide 100mM-2M, sodium laurylsulfonate 5%-20% (weight/volume), hydrochloric acid 0.01M-2.5M;
Described DNA plasmid is in conjunction with the prescription of liquid: Guanidinium hydrochloride 1M-6M, sodium-chlor 10mM-100mM, morpholino ethane sulfonic acid 10mM-100mM, Virahol; Four volume proportions: 40: 40: 10: 10
Described scavenging solution prescription: Trisaminomethane hydrochloric acid 1-50mM adds ethanol 70-85% (volume) again.
2, according to the described plasmid extraction purification kit of claim 1, it is characterized in that, also dispose elutriant, described elutriant is the Trisaminomethane hydrochloric acid of pH7.5-8.5 concentration 8-12mM.
3, a kind of plasmid extraction purification process is characterized in that, may further comprise the steps:
Cracking thalline: 40-100 μ l cracked solution is added 300-1000 μ l (OD600＞2) cultivate bacterium liquid, mix and place 40-80 second;
Pre-treatment DNA column: 150-300 μ l DNA column pre-treatment solution is added in the DNA column, leave heart 8-20 second, reject solution with 10000-15000;
In conjunction with plasmid DNA: 200-400 μ l DNA plasmid binding soln is added among the cracking bacterium liquid, and the centrifugal tubule of overturning is 8-12 time up and down, mixes, and among the DNA column that adding had been handled, leaves heart 10-30 second with 10000-15000;
Clean: add cleaning solution 700-900 μ l, leave heart 8-20 second, reject centrifugate, centrifugal again 50-80 second with 10000-15000;
Wash-out: add 30-40 μ l elutriant in DNA column center, leave heart 20-40 second, can obtain the high purity plasmid DNA with 10000-15000.
4, plasmid extraction purification process according to claim 1 is characterized in that may further comprise the steps:
Cracking thalline: 50 μ l cracked solution are added 500 μ l (OD600＞2) cultivate bacterium liquid, mix and placed for 60 seconds;
Pre-treatment DNA column: 200 μ l DNA column pre-treatment solution are added in the DNA column, left the heart 10 seconds, reject solution with 12000;
In conjunction with plasmid DNA: 300 μ l DNA plasmid binding solns are added among the cracking bacterium liquid, and the centrifugal tubule of overturning is 10 times up and down, mixes, and adds among the DNA column of having handled, and leaves the heart 20 seconds with 12000;
Clean: add cleaning solution 780 μ l, left the heart 10 seconds, reject centrifugate, centrifugal again 60 seconds with 12000.
Wash-out: add 35 μ l elutriants in DNA column center, with 12000 centrifugal 30 seconds.
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| CN 200410047132 CN1274836C (en) | 2004-12-31 | 2004-12-31 | Kit for quick extraction and purification of plasmid and its application |
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| CN 200410047132 CN1274836C (en) | 2004-12-31 | 2004-12-31 | Kit for quick extraction and purification of plasmid and its application |
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| CN1654660A true CN1654660A (en) | 2005-08-17 |
| CN1274836C CN1274836C (en) | 2006-09-13 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103421774A (en) * | 2013-04-26 | 2013-12-04 | 北京师范大学 | Degradative plasmid containing indene [1,2,3-cd] pyrene degradation gene |
| CN108866043A (en) * | 2018-07-17 | 2018-11-23 | 成都医学院 | A kind of pretreatment reagent kit and method of bacterial plasmid extracting |
| CN113512546A (en) * | 2021-04-07 | 2021-10-19 | 湖南中晟全肽生化有限公司 | Rapid plasmid DNA extraction and purification kit and application thereof |
| CN114507662A (en) * | 2022-03-22 | 2022-05-17 | 合肥欧创基因生物科技有限公司 | Binding solution and supercoiled plasmid large-extraction kit based on same |
| CN114958827A (en) * | 2022-06-10 | 2022-08-30 | 通用生物(安徽)股份有限公司 | High-flux plasmid extraction and purification process |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101532011B (en) * | 2008-03-10 | 2010-12-08 | 首都师范大学 | Kit for rapidly extracting and purifying DNA/RNA from same sample and method thereof |
-
2004
- 2004-12-31 CN CN 200410047132 patent/CN1274836C/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103421774A (en) * | 2013-04-26 | 2013-12-04 | 北京师范大学 | Degradative plasmid containing indene [1,2,3-cd] pyrene degradation gene |
| CN108866043A (en) * | 2018-07-17 | 2018-11-23 | 成都医学院 | A kind of pretreatment reagent kit and method of bacterial plasmid extracting |
| CN108866043B (en) * | 2018-07-17 | 2022-02-08 | 成都医学院 | Pretreatment kit and method for bacterial plasmid extraction |
| CN113512546A (en) * | 2021-04-07 | 2021-10-19 | 湖南中晟全肽生化有限公司 | Rapid plasmid DNA extraction and purification kit and application thereof |
| CN114507662A (en) * | 2022-03-22 | 2022-05-17 | 合肥欧创基因生物科技有限公司 | Binding solution and supercoiled plasmid large-extraction kit based on same |
| CN114958827A (en) * | 2022-06-10 | 2022-08-30 | 通用生物(安徽)股份有限公司 | High-flux plasmid extraction and purification process |
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| CN1274836C (en) | 2006-09-13 |
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