CN103205419A - Method and kit for quickly separating and purifying DNA (Deoxyribonucleic Acid) of genome - Google Patents
Method and kit for quickly separating and purifying DNA (Deoxyribonucleic Acid) of genome Download PDFInfo
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- CN103205419A CN103205419A CN2013101471322A CN201310147132A CN103205419A CN 103205419 A CN103205419 A CN 103205419A CN 2013101471322 A CN2013101471322 A CN 2013101471322A CN 201310147132 A CN201310147132 A CN 201310147132A CN 103205419 A CN103205419 A CN 103205419A
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- lysate
- scavenging solution
- magnetic particle
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- tutofusin tris
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Abstract
The invention discloses a method and a kit for quickly separating and purifying the DNA (Deoxyribonucleic Acid) of a genome. The kit comprises a lysis solution, magnetic particles, a cleaning solution and an eluent. The invention provides the method and the kit capable of obtaining high-purity DNA of the genome without heating in the whole process through quick separation and purification by adopting a magnetic particle method; the extraction time is short, the operation is simple, and once-through operation can be completed within 20 minutes; and the sensitivity is high, and samples from 2ul to 1ml can be treated. The purified DNA can be used for downstream experiments such as enzyme digestion, polymerase chain reaction (PCR), library construction and Southern hybridization.
Description
Technical field
The present invention relates to biology field, be particularly related to method and the test kit of the separation and purification that contains sample of nucleic acid, be specifically related to a kind of method and test kit of fast separating and purifying genomic dna.
Background technology
The separation and purification of nucleic acid is the very important basic sample process operation of biology field, and can the quality of DNA be directly connected to subsequent operations and carry out smoothly.Existing various nucleic acid purification methods have liquid phase extraction method and solid phase partition method at present, and in the liquid phase extraction method, nucleic acid maintains in the liquid state, and impurity is removed by centrifugal, precipitation, perhaps nucleic acid is precipitated out by high alcohol; In the solid phase partition method, nucleic acid is touched on the carrier of post by the solid that is combined in of selectivity, and as centrifugal post method, magnetic particle method etc., and impurity is removed by the wash-out of selectivity; More than two classes be universal method, shortcoming such as the liquid phase extraction method exist to be unable to do without the high speed centrifugation operation, operating automation degree is low, extraction yield is not high, parcel impurity is more; Centrifugal post method in the solid phase partition method be unable to do without centrifugal or vacuumizes, and level of automation is low; But the magnetic particle method in the solid phase partition method is the method for the automated operation of comparatively popular approval at present, but the magnetic particle test kit of using at present on the market mostly needs heat temperature raising digestion, wash-out, and during consumption energy consumption, operating process is loaded down with trivial details.
Summary of the invention
The object of the present invention is to provide a kind of method and test kit of fast separating and purifying genomic dna.
One of technical scheme of the present invention:
A kind of test kit of fast separating and purifying genomic dna, described test kit comprises lysate, magnetic particle, scavenging solution and elutriant, described test kit is made up of following reagent:
The described lysate of a comprises lysate I and lysate II; Described lysate I comprises the Proteinase K of 10-70mM Tutofusin tris, 0.1-50mM chelating ion, 50-500mM sodium salt, the negative ion stain remover of 0.2-5%, 0.5-10% neutral detergent and 0.1-0.5mg/ml, the pH value 7-9 of described lysate I; Described lysate II comprises the alcohol of 30-100mM Tutofusin tris, the negative ion stain remover of 0.2-5%, 2-6M guanidinesalt and 10-40%, the pH value 6-7.5 of described lysate II;
The described magnetic particle of b comprises suspension and the magnetic particle of magnetic particle, and described magnetic particle suspension comprises a kind of in two kinds of 5-50mM Tutofusin tris and 50-100% ethanol or the Virahols; Described magnetic particle is the magnetic microsphere of nano level or micron silicon bag quilt, and the magnetic particle in every milliliter the magnetic suspension liquid is 20-100mg;
The described scavenging solution of c comprises scavenging solution I and scavenging solution II; Described scavenging solution I comprises alcohol, 0-10mM Tutofusin tris, 0-200mM Potassium ethanoate and the 0-5% neutral detergent of 0-5M guanidinesalt, 0-50%, the pH value 6-7 of described scavenging solution I; Described scavenging solution II is the alcohol of 10-80%;
The described elutriant of d is the 10-20mM Tutofusin tris, the pH value 7.5-9 of described elutriant.
Preferably: described test kit is made up of following reagent:
The described lysate I of a comprises the Proteinase K of 20mM Tutofusin tris, 10mM disodium ethylene diamine tetraacetate, 100mM sodium-chlor, 2% sodium lauryl sulphate, 4%Triton X-100 and 0.2mg/ml, the pH value 8 of described lysate I;
The described lysate II of b comprises the ethanol of 50mM Tutofusin tris, 0.2% N-sarcosyl, 5M guanidinium isothiocyanate and 20%, the pH value 6.7 of described lysate II;
The described magnetic particle of c comprises 10mM Tutofusin tris and 80% ethanol, and the magnetic particle in every milliliter the magnetic suspension liquid is 50mg;
The described scavenging solution I of d comprises the alcohol of 4M guanidinium isothiocyanate and 30%; Perhaps 200mM Potassium ethanoate, 2%Triton X-100 and 10mM Tutofusin tris, the pH value 6.7 of described scavenging solution I;
The described scavenging solution II of e is 75% ethanol;
The described elutriant of f is the 10mM Tutofusin tris, described elutriant pH value 8.5
Two of technical scheme of the present invention: a kind of method of fast separating and purifying genomic dna, may further comprise the steps: 1, cracking: with blood, seminal fluid, blood sheet, organize equal samples and two kinds of lysates through the digestion of front and back room temperature except Deproteinization, pigment, lipid and other inhibition foreign-matter contaminations, discharge nuclear DNA fully; 2, DNA absorption: at the above-mentioned magnetic particle that in the mixed solution of cracking, adds adsorbable DNA, collect the magnetic particle that has adsorbed DNA through magnetic; 3 clean: the magnetic particle that has adsorbed DNA is cleaned with scavenging solution, after add elutriant room temperature wash-out and go out DNA.
Be specially: namely obtain the high purity genomic dna through lysate room temperature cracking digestion, magnetic particle absorption, scavenging solution cleaning and elutriant room temperature wash-out:
1) gets in the lysate I adding sample of two volumes mixing, room temperature digestion 10min;
2) the lysate II of getting 4 times of sample volumes joins in the sample of above-mentioned digestion, mixing, room temperature digestion 5min;
3) get the 10-30ul magnetic particle and add in the sample, mixing, magnetic is abandoned supernatant;
4) the scavenging solution I of 5-50 times of sample size volume of adding is mixed, and magnetic resolution is abandoned supernatant;
5) the scavenging solution II of 5-50 times of sample size volume of adding is mixed, and magnetic resolution is abandoned supernatant;
6) room temperature is dried, and adds 20-200ul elutriant room temperature wash-out dissolving DNA.
Described cracking digestion and wash-out all are separation and purification DNA at ambient temperature, need not heat.
Described M mole, mM is mmole, described percentage ratio is the described percentage ratio of quality.
Three of technical scheme of the present invention: the purposes of the test kit of described a kind of fast separating and purifying genomic dna in the separation and purification genomic dna.
But the invention provides and a kind ofly need not heat method and the test kit that fast separating and purifying obtains the high purity genomic dna with the magnetic particle whole process; Extraction time is short, and is simple to operate, can finish single job in 20 minutes; Highly sensitive, all can handle to the sample of 1ml greatly to 2ul for a short time.
Description of drawings
The agarose gel electrophoresis figure of Fig. 1 embodiment of the invention 1.
The agarose gel electrophoresis figure of Fig. 2 embodiment of the invention 2.
Embodiment
The present invention is further described by the following examples, yet scope of the present invention is not limited to following embodiment.
Embodiment 1
Test kit reagent is formed:
The lysate I comprises the 20mM Tutofusin tris, 10mM disodium ethylene diamine tetraacetate, 100mM sodium-chlor, 2% sodium lauryl sulphate, 4%Triton X-100, the Proteinase K of 0.2mg/ml, pH value 8.
The lysate II comprises the 50mM Tutofusin tris, 0.2% N-sarcosyl, 5M guanidinium isothiocyanate, 20% ethanol, pH value 6.7.
Magnetic particle comprises the 10mM Tutofusin tris, 80% ethanol, and the magnetic particle in every milliliter the magnetic suspension liquid is 50mg.
Scavenging solution I: 4M guanidinium isothiocyanate and 30% alcohol.
The scavenging solution II is 75% ethanol.
Elutriant: 10mM Tutofusin tris, pH value 8.5.
Separation purification method:
Get 5 parts of anticoagulated whole bloods, every part of 100ul adds in the centrifuge tube of 1.5ml, and the lysate I of getting two volumes adds in the sample, mixing, room temperature digestion 10min.
The lysate II of getting 4 times of sample volumes joins in the sample of above-mentioned digestion, mixing, room temperature digestion 5min.Get an amount of magnetic particle again and add in the sample, mixing, magnetic is abandoned supernatant.
Add 500ul scavenging solution I, mix, magnetic resolution is abandoned supernatant.
Add 500ul scavenging solution II, mix, magnetic resolution is abandoned supernatant.
Room temperature is dried, and adds 50ul elutriant room temperature wash-out dissolving DNA.
Magnetic removes supernatant DNA, gets 5ul respectively and detects with 1% agarose gel electrophoresis, and the result as shown in Figure 1.
Embodiment 2
Test kit reagent is formed:
The lysate I comprises the 20mM Tutofusin tris, 10mM disodium ethylene diamine tetraacetate, 100mM sodium-chlor, 2% sodium lauryl sulphate, 4%Triton X-100, the Proteinase K of 0.2mg/ml, pH value 8.
The lysate II comprises the 50mM Tutofusin tris, 0.2% N-sarcosyl, 5M guanidinium isothiocyanate, 20% ethanol, pH value 6.7.
Magnetic particle comprises the 10mM Tutofusin tris, 80% ethanol, and the magnetic particle in every milliliter the magnetic suspension liquid is 50mg..
Scavenging solution I: 200mM Potassium ethanoate, 2%Triton X-100,10mM Tutofusin tris, pH value 6.7.
The scavenging solution II is 75% ethanol.
Elutriant: 10mM Tutofusin tris, pH value 8.5.
Separation purification method:
Get 3 parts of anticoagulated whole bloods, every part of 100ul adds in the centrifuge tube of 1.5ml, and the lysate I of getting two volumes adds in the sample, mixing, room temperature digestion 10min.
The lysate II of getting 4 times of sample volumes joins in the sample of above-mentioned digestion, mixing, room temperature digestion 5min.Get an amount of magnetic particle again and add in the sample, mixing, magnetic is abandoned supernatant.
Add 1000ul scavenging solution I, mix, magnetic resolution is abandoned supernatant.
Add 1000ul scavenging solution II, mix, magnetic resolution is abandoned supernatant.
Room temperature is dried, and adds 100ul elutriant room temperature wash-out dissolving DNA.
Magnetic removes supernatant DNA, gets 5ul respectively and detects with 1% agarose gel electrophoresis, and the result as shown in Figure 2.
Claims (5)
1. the test kit of a fast separating and purifying genomic dna, described test kit comprises lysate, magnetic particle, scavenging solution and elutriant, it is characterized in that described test kit is made up of following reagent:
The described lysate of a comprises lysate I and lysate II; Described lysate I comprises the Proteinase K of 10-70mM Tutofusin tris, 0.1-50mM chelating ion, 50-500mM sodium salt, the negative ion stain remover of 0.2-5%, 0.5-10% neutral detergent and 0.1-0.5mg/ml, the pH value 7-9 of described lysate I; Described lysate II comprises the alcohol of 30-100mM Tutofusin tris, the negative ion stain remover of 0.2-5%, 2-6M guanidinesalt and 10-40%, the pH value 6-7.5 of described lysate II;
The described magnetic particle of b comprises suspension and the magnetic particle of magnetic particle, and described magnetic particle suspension comprises a kind of in two kinds of 5-50mM Tutofusin tris and 50-100% ethanol or the Virahols; Described magnetic particle is the magnetic microsphere of nano level or micron silicon bag quilt, and the magnetic particle in every milliliter the magnetic suspension liquid is 20-100mg;
The described scavenging solution of c comprises scavenging solution I and scavenging solution II; Described scavenging solution I comprises alcohol, 0-10mM Tutofusin tris, 0-200mM Potassium ethanoate and the 0-5% neutral detergent of 0-5M guanidinesalt, 0-50%, the pH value 6-7 of described scavenging solution I; Described scavenging solution II is the alcohol of 10-80%;
The described elutriant of d is the 10-20mM Tutofusin tris, the pH value 7.5-9 of described elutriant.
2. the test kit of a kind of fast separating and purifying genomic dna according to claim 1 is characterized in that described test kit is made up of following reagent:
The described lysate I of a comprises the Proteinase K of 20mM Tutofusin tris, 10mM disodium ethylene diamine tetraacetate, 100mM sodium-chlor, 2% sodium lauryl sulphate, 4%Triton X-100 and 0.2mg/ml, the pH value 8 of described lysate I;
The described lysate II of b comprises the ethanol of 50mM Tutofusin tris, 0.2% N-sarcosyl, 5M guanidinium isothiocyanate and 20%, the pH value 6.7 of described lysate II;
The described magnetic particle of c comprises 10mM Tutofusin tris and 80% ethanol, and the magnetic particle in every milliliter the magnetic suspension liquid is 50mg;
The described scavenging solution I of d comprises the alcohol of 4M guanidinium isothiocyanate and 30%; Perhaps 200mM Potassium ethanoate, 2%Triton X-100 and 10mM Tutofusin tris, the pH value 6.7 of described scavenging solution I;
The described scavenging solution II of e is 75% ethanol;
The described elutriant of f is the 10mM Tutofusin tris, described elutriant pH value 8.5.
3. the method for a fast separating and purifying genomic dna, utilize the test kit of claim 1 or 2 described a kind of fast separating and purifying genomic dnas, namely obtain the high purity genomic dna through lysate room temperature cracking digestion, magnetic particle absorption, scavenging solution cleaning and elutriant room temperature wash-out, it is characterized in that method is as follows:
1) gets in the lysate I adding sample of two volumes mixing, room temperature digestion 10min;
2) the lysate II of getting 4 times of sample volumes joins in the sample of above-mentioned digestion, mixing, room temperature digestion 5min;
3) get the 10-30ul magnetic particle and add in the sample, mixing, magnetic is abandoned supernatant;
4) the scavenging solution I of 5-50 times of sample size volume of adding is mixed, and magnetic resolution is abandoned supernatant;
5) the scavenging solution II of 5-50 times of sample size volume of adding is mixed, and magnetic resolution is abandoned supernatant;
6) room temperature is dried, and adds 20-200ul elutriant room temperature wash-out dissolving DNA.
4. the method for a kind of fast separating and purifying genomic dna according to claim 3 is characterized in that described cracking digestion and wash-out all are separation and purification DNA at ambient temperature.
5. the purposes of test kit in the separation and purification genomic dna of claim 1 or 2 described a kind of fast separating and purifying genomic dnas.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2013101471322A CN103205419A (en) | 2013-04-25 | 2013-04-25 | Method and kit for quickly separating and purifying DNA (Deoxyribonucleic Acid) of genome |
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| CN2013101471322A CN103205419A (en) | 2013-04-25 | 2013-04-25 | Method and kit for quickly separating and purifying DNA (Deoxyribonucleic Acid) of genome |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107151666A (en) * | 2016-03-03 | 2017-09-12 | 上海市农业科学院 | The extracting method of microbial DNA in a kind of water body |
| CN109694863A (en) * | 2019-02-28 | 2019-04-30 | 深圳市刚竹医疗科技有限公司 | Extracting method for the lysate of nucleic acid extraction, cleaning solution, nucleic acid extraction kit and nucleic acid |
| CN109913527A (en) * | 2019-02-11 | 2019-06-21 | 张丽英 | A method of utilizing shigella dysenteriae in ATP bioluminescence reaction detection food |
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| CN101812444A (en) * | 2010-04-23 | 2010-08-25 | 北京博迈世纪生物技术有限公司 | Blood genome magnetic bead small-amount extraction reagent kit and extraction method thereof |
| CN101935645A (en) * | 2010-09-13 | 2011-01-05 | 原平皓(天津)生物技术有限公司 | Kit for extracting DNA from histiocytes and method thereof |
| CN102888397A (en) * | 2012-09-25 | 2013-01-23 | 杭州硕航生物科技有限公司 | Kit using magnetic bead to extract whole blood genomic DNA and use of kit |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101812444A (en) * | 2010-04-23 | 2010-08-25 | 北京博迈世纪生物技术有限公司 | Blood genome magnetic bead small-amount extraction reagent kit and extraction method thereof |
| CN101935645A (en) * | 2010-09-13 | 2011-01-05 | 原平皓(天津)生物技术有限公司 | Kit for extracting DNA from histiocytes and method thereof |
| CN102888397A (en) * | 2012-09-25 | 2013-01-23 | 杭州硕航生物科技有限公司 | Kit using magnetic bead to extract whole blood genomic DNA and use of kit |
Non-Patent Citations (1)
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| 卢瑛等: "一种快速提取基因组DNA的方法", 《中国生物工程杂志》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107151666A (en) * | 2016-03-03 | 2017-09-12 | 上海市农业科学院 | The extracting method of microbial DNA in a kind of water body |
| CN109913527A (en) * | 2019-02-11 | 2019-06-21 | 张丽英 | A method of utilizing shigella dysenteriae in ATP bioluminescence reaction detection food |
| CN109694863A (en) * | 2019-02-28 | 2019-04-30 | 深圳市刚竹医疗科技有限公司 | Extracting method for the lysate of nucleic acid extraction, cleaning solution, nucleic acid extraction kit and nucleic acid |
| CN109694863B (en) * | 2019-02-28 | 2021-04-20 | 深圳市刚竹医疗科技有限公司 | Lysis solution for nucleic acid extraction, cleaning solution, nucleic acid extraction kit and nucleic acid extraction method |
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Application publication date: 20130717 |