CN1226600A - Reagent box for extracting DNA fast and use thereof - Google Patents
Reagent box for extracting DNA fast and use thereof Download PDFInfo
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- CN1226600A CN1226600A CN 99113448 CN99113448A CN1226600A CN 1226600 A CN1226600 A CN 1226600A CN 99113448 CN99113448 CN 99113448 CN 99113448 A CN99113448 A CN 99113448A CN 1226600 A CN1226600 A CN 1226600A
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Abstract
A reagent box used to quickly extract the DNA from plasmids contains 6 reagents: buffer liquid for bacterial suspension, buffer liquid for bacterial cracking, buffer liquid for plasmid precipitation, isopropanol, buffer liquid for protein precipitation and DNA diluent. Its advantages include high-speed,simple, efficient, cheap and high-quality extraction, no harm, and clear electrophoresis strip of DNA (1A, 2A, 3A) without RNA impurities.
Description
The present invention relates to the molecular biology research field, is a kind of test kit and application method thereof of simple and easy rapid extraction plasmid DNA.
The extraction of plasmid DNA and purifying are to be most widely used in the molecular biology research field and the most basic technology.Therefore, main both at home and abroad biological reagent company has all developed the relevant test kit of technology therewith, as bio-engineering corporations such as Qiagen, Promega, magnificent, Hua Shun, brilliant U.S.s.External test kit all is based on purification column, thereby the cost height, costs an arm and a leg.Domestic test kit mainly is the improvement of being done on traditional alkaline lysis, the extractive basis of phenol-chloroform, its plasmid DNA of obtaining must be measured low, of low quality, can not satisfy the requirement of connection in the molecular biology experiment, sequencing and transfection, leaching process length consuming time, and, peculiar smell is arranged because of phenol and chloroform are toxic substance, both contaminate environment endangered experiment operator health again.
The test kit and the application method thereof that the purpose of this invention is to provide a kind of quick, simple and easy, efficient, high-quality, inexpensive, harmless extraction plasmid DNA.
In the plasmid DNA extractive technique, obtain the plasmid DNA that quality is good, purity is high, gordian technique is the elimination of cellular lysate, protein and RNA.Core of the present invention is the salt that agents useful for same adopts different pH and different concns, remove protein and RNA with step-by-step precipitation method substituted phenol-chloroform extraction method, the plasmid DNA purity of being extracted can satisfy the relevant experimental requirements of molecular biology, comprises that the clone identifies, enzyme is cut, connection, sequencing and transfection etc.The present invention includes 6 kinds of reagent, i.e. the resuspended damping fluid of thalline (R1); Cellular lysate damping fluid (R2); Plasmid precipitation buffering liquid (R3); Virahol (R4); Albumen precipitation damping fluid (R5); DNA diluent (R6).Their composition and proportioning are respectively:
Reagent R1:
Component: 50mmol glucose
25mmol?Tris-Hcl(pH8.0)
10mmol?EDTA(pH8.0)
The 5mg/ml N,O-Diacetylmuramidase
Proportioning: preparation 100ml R1
Get glucose (COH
12O
6.H
2O) 0.991 gram
Distilled water 80ml
0.5mol?EDTA(pH8.0) 2ml
1mol?Tris-Hcl(pH8.0)2.5ml
Be settled to 100ml with distilled water, 10Ibf/in
2(6.895 * 10
4Pa) autoclaving is 15 minutes, adds the 500mg N,O-Diacetylmuramidase again.Press 25 usage quantity packing 5.6ml in the opaque-plastic bottle, 4 ℃ of preservations.
Reagent R2:
Component: 0.2mol NaOH
1%SDS
Proportioning: preparation 100ml R2
Get 10mol NaOH 2ml
Distilled water 80ml
10%SDS 10ml
Be settled to 100ml with distilled water, press 25 usage quantity packing 11.2ml in opaque-plastic bottle, room temperature preservation.
Reagent R3:
Component: 5.0~6.0mol ammonium acetate (or sodium-acetate), best ammonium acetate (or sodium-acetate) concentration is 5.5mol.
Proportioning: preparation 100ml R3
Get ammonium acetate 42.4g
Distilled water 80ml
After the stirring and dissolving, transfer to pH7.4~7.6, its preferred pH value 7.5 to 100ml, is pressed 25 usage quantity packing 8.4ml in opaque-plastic bottle, room temperature preservation with the distilled water constant volume.
Reagent R4: Virahol (analytical pure) is a commercialization reagent.
Reagent R5:
Component: 1.2~1.6mol ammonium acetate (or sodium-acetate), best ammonium acetate (or sodium-acetate) concentration is 1.4mol.
Proportioning: preparation 100ml R5
Get ammonium acetate 10.8g
Distilled water 80ml
After the stirring and dissolving, transfer to pH7.2~7.3, its preferred pH value is 7.25.Be settled to 100ml with distilled water, press 25 usage quantity packing 2.8ml in opaque-plastic bottle, room temperature preservation.
Reagent R6:
Component: TE damping fluid (pH7.6) 100ml
RNase?A 10mg
RNase T 10,000 ten thousand units
Proportioning: preparation 100ml TE damping fluid
Get Tris salt (analytical pure) 1.58g
EDTA (analytical pure) 0.372g
Distilled water 80ml
After the stirring and dissolving, transfer to pH7.6, be settled to 100ml, add RNase A 10mg and RNase T 10,000 ten thousand units, press 25 usage quantity packing 0.56ml in the opaque-plastic bottle, 4 ℃ of preservations with distilled water.
The main advantage of the present invention is: easy and simple to handle, the result is stable; Weak point consuming time can be finished all processes about 80 minutes; Must measure height, to a high copy number plasmid, the conventional cultivation 16 hours is as bacterial growth density OD
600Be about at 3.5 o'clock, 1.5ml bacterium liquid can obtain 10 μ g~15 μ g DNA; Purity is good, and no protein and RNA pollute, and the plasmid DNA of extraction can supply endonuclease digestion, connection, sequencing, eukaryotic cell transfection equimolecular biological experiment to use; Inexpensive, compare with similar products at home and abroad, price reduces by 20%~40%; Owing to need not use phenol and chloroform, both eliminated the worry of environmental pollution, harmless to the experiment operator health again.
Using this test kit extraction plasmid DNA can operate by following method:
1. shake bacterium amplification plasmid (seeing " molecular cloning experiment guide " for details) routinely;
2. get conventional cultivation 16 hours, bacterial growth density OD
600Be about 3.5 bacterium liquid 1.5ml in the 1.5ml centrifuge tube,
Room temperature is abandoned supernatant with 12000rpm centrifugal 30 seconds;
3. add 200 μ l R1 in the precipitation, mixing fully vibrates;
4. add 400 μ l R2, put upside down 5 rearmounted ice baths gently 2 minutes;
5. the 300 μ l R3 that add precooling put upside down 5 rearmounted ice baths 2 minutes gently;
6. room temperature is with 12000rpm centrifugal 5 minutes;
7. the collection supernatant added the rearmounted room temperature of 600 μ l R4 mixings 2 minutes;
8. room temperature is with 12000rpm centrifugal 10 minutes;
9. abandon supernatant, added the rearmounted ice bath of the resuspended precipitation of 100 μ l R5 immediately 2 minutes;
10. room temperature is with 12000rpm centrifugal 5 minutes;
11. the collection supernatant adds 100 μ l R4 mixings;
12. room temperature centrifugal 5 minutes with 12000rpm is abandoned supernatant, precipitation is washed with 75% ethanol, is dissolved in 20 μ l R6 after the drying,
37 ℃ of water-baths 15 minutes;
13. add 10 μ l R3 and 30 μ l R4 mixings, room temperature 2 minutes;
14. room temperature centrifugal 10 minutes with 12000rpm is abandoned supernatant;
15. precipitation is washed with 75% ethanol, is dissolved in 15 μ l TE damping fluids after the drying ,-20 ℃ of preservations are standby;
The gained plasmid DNA can be verified with 0.8% agarose gel electrophoresis.
Fig. 1 shows the plasmid DNA electrophoresis contrast collection of illustrative plates of the thalline that contains same plasmid DNA being used this test kit and the extraction of application Qiagen pillar purification kit.Among the figure, M is a λ DNA/Hind III molecular weight marker, and 1A, 2A, 3A are the plasmid DNA that this test kit of use is extracted, 1B, 2B, the corresponding plasmid DNA of 3B for using Qiagen pillar purification cassette to be extracted.Use the plasmid DNA of two kinds of test kit gained as seen from the figure, electrophoretic band is clear, and no RNA mixes.Illustrate and use the present invention plasmid DNA of extracting and the same plasmid DNA of using Qiagen pillar purification kit to extract relatively, in purity with must not have obvious difference aspect the amount.
Claims (6)
1. test kit that extracts plasmid DNA, it is characterized in that comprising the resuspended damping fluid of thalline (R1), cellular lysate damping fluid (R2), plasmid precipitation buffering liquid (R3), Virahol (R4), albumen precipitation damping fluid (R5) and six kinds of reagent of DNA diluent (R6), the component of R1 is a 50mmol glucose, 25mmol Tris-Hcl (pH8.0), 10mmol EDTA (pH8.0), the 5mg/ml N,O-Diacetylmuramidase; The component of R2 is 0.2mol NaOH, 1%SDS; The component of R3 is 5.0~6.0mol ammonium acetate (or sodium-acetate), pH7.4~7.6; R4 is analytically pure Virahol; The component of R5 is 1.2~1.6mol ammonium acetate (or sodium-acetate), pH7.2~7.3; The component of R6 is that the adding final concentration is 0.1mg/ml RNase A and 100 units/mlRNase T in the TE damping fluid.
2. by the test kit of the described extraction plasmid DNA of claim 1, the optimum concn that it is characterized in that described reagent R3 is 5.5mol ammonium acetate (or sodium-acetate).
3. by the test kit of the described extraction plasmid DNA of claim 1, the optimal ph that it is characterized in that reagent R3 is 7.5.
4. by the test kit of the described extraction plasmid DNA of claim 1, the optimum concn that it is characterized in that reagent R5 is 1.4mol ammonium acetate (or sodium-acetate).
5. by the test kit of the described extraction plasmid DNA of claim 1, it is characterized in that reagent R5 optimal ph is 7.25.
6. the application method of the described extraction plasmid DNA of claim 1 test kit is characterized in that operating as follows:
(1) shakes bacterium amplification plasmid (seeing " molecular cloning experiment guide " for details) routinely;
(2) learn from else's experience and conventional cultivated bacterial growth density OD 16 hours
600About 3.5 bacterium liquid 1.5ml is in the 1.5ml centrifuge tube,
Room temperature is abandoned supernatant with 12000rpm centrifugal 30 seconds;
(3) add 200 μ l R1 in the precipitation, mixing fully vibrates;
(4) add 400 μ l R2, put upside down 5 rearmounted ice baths gently 2 minutes;
(5) add 300 μ l R3 of precooling, put upside down 5 rearmounted ice baths gently 2 minutes;
(6) room temperature is with 12000rpm centrifugal 5 minutes;
(7) collect supernatant, added the rearmounted room temperature of 600 μ l R4 mixings 2 minutes;
(8) room temperature is with 12000rpm centrifugal 10 minutes;
(9) abandon supernatant, added the rearmounted ice bath of the resuspended precipitation of 100 μ l R5 immediately 2 minutes;
(10) room temperature is with 12000rpm centrifugal 5 minutes;
(11) collect supernatant, add 100 μ l R4 mixings;
(12) room temperature is with 12000rpm centrifugal 5 minutes, abandons supernatant, and precipitation is washed with 75% ethanol, is dissolved in 20 μ l R6 after the drying, 37 ℃ of water-baths 15 minutes;
(13) add 10 μ l R3 and 30 μ l R4 mixings, room temperature 2 minutes;
(14) room temperature is with 12000rpm centrifugal 10 minutes, abandons supernatant;
(15) precipitation is washed with 75% ethanol, is dissolved in 15 μ l TE damping fluids after the drying, and-20 ℃ of preservations are standby;
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CN99113448A CN1086201C (en) | 1999-01-29 | 1999-01-29 | Reagent box for extracting DNA fast and use thereof |
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CN99113448A CN1086201C (en) | 1999-01-29 | 1999-01-29 | Reagent box for extracting DNA fast and use thereof |
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CN1086201C CN1086201C (en) | 2002-06-12 |
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Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN100368539C (en) * | 2006-11-10 | 2008-02-13 | 中南大学 | Method for extracting high molecular genome from bacterium |
CN101235379B (en) * | 2007-02-02 | 2010-06-02 | 中国科学院沈阳应用生态研究所 | Method for extracting bacteria plasmid DNA |
CN101338312B (en) * | 2008-07-03 | 2011-03-23 | 天根生化科技(北京)有限公司 | Pellosil activate fluid and applications thereof for extracting nucleic acid |
CN102206627A (en) * | 2011-03-18 | 2011-10-05 | 青岛大学医学院附属医院 | Reagent and method for extracting plasmid DNA |
CN106867994A (en) * | 2017-02-27 | 2017-06-20 | 天根生化科技(北京)有限公司 | It is applied to rapid precipitation buffer solution and its application of extraction of plasmid DNA |
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 |
CN117969228A (en) * | 2024-04-02 | 2024-05-03 | 成都翼泰生物科技有限公司 | Bacterial liquid diluent, preparation method, reference, kit and application thereof |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
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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 |
-
1999
- 1999-01-29 CN CN99113448A patent/CN1086201C/en not_active Expired - Fee Related
Cited By (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN100368539C (en) * | 2006-11-10 | 2008-02-13 | 中南大学 | Method for extracting high molecular genome from bacterium |
CN101235379B (en) * | 2007-02-02 | 2010-06-02 | 中国科学院沈阳应用生态研究所 | Method for extracting bacteria plasmid DNA |
CN101338312B (en) * | 2008-07-03 | 2011-03-23 | 天根生化科技(北京)有限公司 | Pellosil activate fluid and applications thereof for extracting nucleic acid |
CN102206627A (en) * | 2011-03-18 | 2011-10-05 | 青岛大学医学院附属医院 | Reagent and method for extracting plasmid DNA |
CN102206627B (en) * | 2011-03-18 | 2012-11-14 | 青岛大学医学院附属医院 | Reagent and method for extracting plasmid DNA |
CN106867994A (en) * | 2017-02-27 | 2017-06-20 | 天根生化科技(北京)有限公司 | It is applied to rapid precipitation buffer solution and its application of extraction of plasmid DNA |
CN106867994B (en) * | 2017-02-27 | 2020-10-30 | 天根生化科技(北京)有限公司 | Rapid precipitation buffer solution applied to plasmid DNA extraction and application thereof |
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 |
CN117969228A (en) * | 2024-04-02 | 2024-05-03 | 成都翼泰生物科技有限公司 | Bacterial liquid diluent, preparation method, reference, kit and application thereof |
CN117969228B (en) * | 2024-04-02 | 2024-05-28 | 成都翼泰生物科技有限公司 | Bacterial liquid diluent, preparation method, reference, kit and application thereof |
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