CN104845963A - Method for high flux rapid extraction of vegetable single seed DNA - Google Patents
Method for high flux rapid extraction of vegetable single seed DNA Download PDFInfo
- Publication number
- CN104845963A CN104845963A CN201410051556.3A CN201410051556A CN104845963A CN 104845963 A CN104845963 A CN 104845963A CN 201410051556 A CN201410051556 A CN 201410051556A CN 104845963 A CN104845963 A CN 104845963A
- Authority
- CN
- China
- Prior art keywords
- dna
- sample
- add
- chinese cabbage
- extraction
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for high flux rapid extraction of vegetable single seed DNA, an early seedling single plant which is developed from a seed by breaking of hull, a to-be-tested sample single plant is ground into powder in a 1.5 mL pointed bottom centrifugal tube by a handheld electric disruptor, then100 mu L of 10 % Chelex-100 solution is added, the mixture is in water bath at 65 DEG C for 20 min, the mixture is quickly frozen for 10 min, and centrifuged for 3 min ate the speed of 13200 RPM/min, the supernatant is sucked to obtain sample DNA, and the sample DNA can be directly used for PCR amplification. According to the DNA extraction method, the whole course of sample DNA extraction can be finished in 35 minutes. The extracted DNA quality standards meet the requirements of the subsequent PCR amplification, the method is more suitable for the purpose of breeding industry variety breeding, high flux rapid extraction of DNA in the sample, and has the advantages of being efficient, fast, low in cost, simple in operation and the like.
Description
Technical field
The invention belongs to technical field of molecular biology, relate to the preparation method of template DNA, is a kind of efficient, quick, low cost, high-throughput vegetables single seed DNA extraction method easy and simple to handle.
Background technology
Protocols in Molecular Biology has penetrated into all respects such as biology, medical science, phytology, genetics and zoology.The extraction of DNA of plants is the most basic step of carrying out analysis of molecules, in plant genetic engineering and molecular biology of plants research, occupy critical role, and the quality of the DNA quality extracted will be directly connected to the success or failure of later stage molecular biology test research.The molecule marker of PCR-based technology applies to Purity Identification, the analysis of sibship, molecular mark, the assignment of genes gene mapping and transfer-gen plant detection etc. widely.Mark wherein based on round pcr is as simple sequence repeat marker (SSR marker or microsatellite marker), due to its have that polymorphism is good, reliability is high, technology is simple, low price and the advantage such as DNA profiling usage quantity is low, be widely used in molecular biology.For a long time, in single seed sample, the extraction of DNA is consuming time, loaded down with trivial details process always, and seriously slowed down detection speed.Therefore, many scholars are exploring the high-throughput rapid extracting method of vegetables single seed DNA always.
Research both at home and abroad at present finds and the DNA extraction method used has a lot, mainly contains Traditional Method, resin method, paramagnetic particle method, immune affinity method etc.
(1) Traditional Method is by CTAB(cetyl trimethylammonium bromide) or SDS(sodium lauryl sulphate) lysing cell, the organic solvent extract proteins such as phenol/chloroform, can the precipitation of Polysaccharide removing and aldehydes matter effectively, thus be applicable to extract DNA from containing phenols and the higher vegetable material of glucide, the DNA purity obtained is high, and content is many.But more time-consuming, need 5 hours even more, flow process is loaded down with trivial details, and simultaneously sample needs 4-5 EP to manage, and easily causes and obscures and pollute, complicated operation.And in leaching process, employ a large amount of organic solvents, damage the health of experimenter.
(2) paramagnetic particle method extracts DNA by magnetic bead absorption, magnetic field separation, and be applicable to freezing, outmoded tissue, simple, quick whole process only need, less than 2 hours, utilize magnetic field separation can obtain purer DNA, but output than Traditional Method obtain few, and cost is higher.
(3) immune affinity method extracts DNA by antigen antibody reaction, is applicable to the sample that sample content is little, and the DNA purity of acquisition is high, and content is many.Shortcoming is that equipment requirements is higher, not easily popularizes, and the preparation of anti-DNA single clonal antibody is committed step.
(4) resin method uses Chelex-100 resin to extract DNA, it is reported the method at present for bacterium, blood and fractionated viral nucleic acid extraction, simple to operate, quick and cost is not high.Avoid using the organic solvents such as phenol chloroform simultaneously, operator are not damaged.
Chelex-100 is a kind of chemical finishing resin, is made up of vinylbenzene, divinylbenzene interpolymer.Containing paired Iminodiacetate ion, integrate polyvalent metal ion, especially selectivity integrates divalent ion, has higher metalloform-selective and stronger bonding force than conventional ion exchanger. and can in conjunction with many other allogenic materials that may affect a step and analyze.By centrifugal removing Chelex-100 particle, the material that these and Chelex-100 are combined is separated with DNA, prevent from being attached to inhibitor in Chelex or impurity takes in PCR reaction, affect next step DNA analysis, and pass through bind metal ion, prevent DNA degradation, and improve pcr amplification success ratio.Singer.Sam in 1989 etc. have reported at first and have extracted tissue culture cells DNA by Chelex-l00 method, the systematic researches such as Walsh in 1991 embody rule of the method in multiple legal medical expert's biological material.Current Chelex-100 method expands to Other subjects gradually, the DNA extracted by this method is easy and simple to handle, and the loss of DNA in leaching process can be reduced, be usually used in extracting enough DNA in (as medium in hair, blood, seminal stain and exuviation cell) in very micro-sample.The present invention utilizes Chelex-100 to carry out high-throughput rapid extraction to the fresh individual plant sample that single seed broken shell is developed to initial stage seedling, improves the detection efficiency of breeding industries breed breeding and Purity process.
Summary of the invention
The object of the invention is to overcome the shortcomings such as traditional sample DNA extraction step is many, complicated, time-consuming, disclose a kind of method of high-throughput rapid extraction vegetables single seed DNA.DNA extraction method of the present invention has efficiently, fast, low cost, the advantage such as easy and simple to handle.
For achieving the above object, the invention provides following technical scheme:
A method of high-throughput rapid extraction vegetables single seed DNA, is characterized in that it carries out as follows:
(1) get plant sample 100mg to be measured to add in 1.5mL sterile centrifugation tube, utilize the broken instrument of hand electric to be ground to powder;
(2) add 100 μ L10%Chelex-100 solution, vortex fully mixes, and is placed in 65 DEG C of water-bath water bath with thermostatic control 20min;
(3) rapid freezing 10min, 13200rpm/min on ice after water-bath, centrifugal 3min, gets supernatant, preserves, for subsequent PCR amplification.
Sample to be tested of the present invention is that plant sample comprises all kinds of vegetable plant samples such as Cauliflower, Chinese cabbage, cucumber, celery.Sample to be tested of the present invention is the fresh individual plant sample that plant seed broken shell is developed to initial stage seedling.
Chelex-100 strength of solution of the present invention is 10%, and add-on is 100 μ L; Water bath with thermostatic control temperature is 65 DEG C, and the time is 20min; Centrifugal speed is 13200rpm/min, and the time is 3min.
In order to can explanation extracting method of the present invention clearly, be sample instance below with Chinese cabbage, to the in addition compare explanation respectively of extracting method of the present invention and traditional CTAB method.
One, materials and methods
(1) sample: cabbage hybrid, is provided by Tianjin Ke Run Vegetable Research Institute.
(2) key instrument: high speed freezing centrifuge, PCR amplification instrument, thermostat water bath, gel electrophoresis equipment.
(3) main agents:
EST-PCR primer is synthesized by Shanghai Sheng Gong company, and primer sequence is in table 1.
2 ×
master Mixes reagent is purchased from Promega company.
Chelex-100 is purchased from sigma company.
10%Chelex-100 solution: weigh 10g Chelex-100, add 90mL deionized water, 121 DEG C of sterilizings, the used time mixes.
CTAB(cetyl trimethylammonium bromide) Extraction buffer: dissolve CTAB20g in 1000mL water, Tris12.11g, NaCl81.82g, Na2EDTA7.44g, autoclaving.
Table 1 Chinese cabbage Purity EST-SSR primer sequence
Two, the comparison of two kinds of extracting method of cabbage hybrid DNA
1, CTAB method extracts DNA
(1) get cabbage hybrid individual plant seedling and be about 100mg, utilize the broken instrument of hand electric to be ground to powder;
(2) add the CTAB Extraction buffer that 800 μ L are preheated to 65 DEG C, fully mix, 65 DEG C of water-bath 60min, period does not stop to put upside down mixing;
(3) the centrifugal 10min of 13200rpm/min, the new centrifuge tube of transfer supernatant to, adds the chloroform isoamyl alcohol of 500 μ L, fully mixes, the centrifugal 10min of 13200rpm/min, the new centrifuge tube of transfer supernatant to; Repeat 1 time;
(4) add the CTAB precipitation buffering liquid of 2 times of volumes, mixing, room temperature leaves standstill 60min; The centrifugal 10min of 13200rpm/min, abandons supernatant;
(5) add 350 μ L sodium chloride solution dissolution precipitations in precipitation, then add 350 μ L chloroform isoamyl alcohols, fully mix, the centrifugal 10min of 13200rpm/min, the new centrifuge tube of transfer supernatant to;
(6) add 0.6 times of volume isopropanol, mixing, the centrifugal 10min of 13200rpm/min, abandons supernatant;
(7) add 500 μ L70% ethanolic solns in precipitation, the centrifugal 10min of 13200rpm/min, abandons supernatant; Repeat 1 time; Drying precipitated, add 100 μ L sterilizing deionized water dissolving DNA.
2, the inventive method extracts DNA
(1) get cabbage hybrid individual plant seedling to be about 100mg and to add in 1.5mL sterile centrifugation tube, utilize the broken instrument of hand electric to be ground to powder;
(2) add 100 μ L10%Chelex-100 solution, vortex fully mixes, and is placed in 65 DEG C of water-bath water bath with thermostatic control 20min;
(3) rapid freezing 10min, 13200rpm/min on ice after water-bath, centrifugal 3min, gets supernatant, preserves, for subsequent PCR amplification.
Three, the pcr amplification detection experiment of two kinds of method extraction DNA:
1, PCR reaction system composition:
2 ×
the each 0.25 μ L of Master Mixes5 μ L, upstream and downstream primer 10 μm of ol/L, experimental cultivar Chinese cabbage genomic DNA template, concentration 50ng/ μ L, 1 μ L, distilled water supplies 10 μ L;
2, PCR response procedures:
94 DEG C of denaturation 5min, 35 amplification cycles (94 DEG C of 1min, 53.5 DEG C of 45sec, 72 DEG C of 45sec); 72 DEG C extend 7min, 4 DEG C of preservations;
3, Product Identification: 8% non denatured polyacrylate hydrogel electrophoresis
Deposition condition, voltage: 250V; Time: 30min.
Silver dye is observed, and does molecular weight marker with 20bp Marker.
4, pcr amplification result:
To the DNA that two kinds of methods are extracted, carry out the amplification of Chinese cabbage Purity primer BC1, BC9, BC26 respectively, non denatured polyacrylate hydrogel electrophoresis result is shown in accompanying drawing.Result shows, and the amplification of two kinds of method extraction DNA is basically identical.
The positively effect that the present invention has compared with conventional art is:
(1) traditional DNA extraction operation steps is many, complicated, time-consuming.Need through multiple step such as cracking, extracting, need mutually to shift sample simultaneously between multiple centrifuge tube, add the possibility to crossed contamination between sample.In addition, the organic solvent such as chloroform is harmful.And easy, the whole leaching process of DNA extraction method process of the present invention can complete in same centrifuge tube, simultaneously without the need to using toxic organic solvents, all can not work the mischief to human body and environment.
(2) advantage that present method is maximum is that its process is quick, easy, substantially increases the detection efficiency of breeding industries cultivar identification and Purity.Compared with needing 5 hours with traditional leaching process, adopt present method, 35min just can complete the whole process that sample DNA extracts.
(3) DNA adopting the method for rapid extraction sample DNA of the present invention to obtain is through PCR test experience, confirm that the DNA obtained with traditional method is basically identical, this method can be adopted completely to substitute complexity, time-consuming traditional CT AB method, thus greatly save the time.
Accompanying drawing illustrates:
Fig. 1 is the electrophoretic analysis collection of illustrative plates of EST-SSR primer BC1 for Chinese cabbage cultivar amplified production; Be followed successively by Chinese cabbage cultivar " autumn green 75 ", Chinese cabbage cultivar " Tianjin autumn 78 " (traditional CT AB method) from left to right; Chinese cabbage cultivar " autumn green 75 ", Chinese cabbage cultivar " Tianjin autumn 78 " (the inventive method);
Fig. 2 is the electrophoretic analysis collection of illustrative plates of EST-SSR primer BC9 for Chinese cabbage cultivar amplified production; Be followed successively by Chinese cabbage cultivar " autumn green 75 ", Chinese cabbage cultivar " autumn green 60 " (traditional CT AB method) from left to right; Chinese cabbage cultivar " autumn green 75 ", Chinese cabbage cultivar " autumn green 60 " (the inventive method);
Fig. 3 is the electrophoretic analysis collection of illustrative plates of EST-SSR primer BC26 for Chinese cabbage cultivar amplified production; Be followed successively by Chinese cabbage cultivar " autumn green 75 ", Chinese cabbage cultivar " Tianjin autumn 78 " (traditional CT AB method) from left to right; Chinese cabbage cultivar " autumn green 75 ", Chinese cabbage cultivar " Tianjin autumn 78 " (the inventive method);
Fig. 4 is the electrophoretic analysis collection of illustrative plates of Cauliflower EST-SSR primer HYC33 amplified production; Being followed successively by 1-5 is from left to right the maternal O5H2 amplified production that the inventive method extracts DNA; 6-25 is 20 strains " Tianjin product 70 " the F1 generation cross-fertilize seed Cauliflower amplification that the inventive method extracts DNA; 26-30 is male parent G × H amplification that the inventive method extracts DNA.
Fig. 5 is the electrophoretic analysis collection of illustrative plates of Chinese cabbage EST-SSR primer BC1 amplified production; Being followed successively by M is from left to right 20bpMarker; 1-43 is 43 strain Chinese cabbage cultivars " autumn green 75 " the cross-fertilize seed amplification that the inventive method extracts DNA.
Fig. 6 is the electrophoretic analysis collection of illustrative plates of Chinese cabbage EST-SSR primer BC9 amplified production; Being followed successively by M is from left to right 20bpMarker; 1-37 is 37 strain Chinese cabbage cultivars " Tianjin white 56 " the cross-fertilize seed amplification that the inventive method extracts DNA.
Fig. 7 is the electrophoretic analysis collection of illustrative plates of Chinese cabbage EST-SSR primer BC9 amplified production; Being followed successively by M is from left to right 20bpMarker; 1-46 is 46 strain Chinese cabbage cultivars " autumn green 60 " the cross-fertilize seed amplification that the inventive method extracts DNA.
Embodiment
In order to can explanation method of the present invention clearly, below in conjunction with embodiment, the present invention will be further described.
Embodiment 1
Be sample for examination cauliflower variety " Tianjin product 70 " cross-fertilize seed, adopt the inventive method DNA rapid extraction.Preparation method is as follows:
(1) get Cauliflower cross-fertilize seed individual plant seedling to be about 100mg and to add in 1.5mL sterile centrifugation tube, utilize the broken instrument of hand electric to be ground to powder;
(2) add 100 μ L10%Chelex-100 solution, vortex fully mixes, and is placed in 65 DEG C of water-bath water bath with thermostatic control 20min;
(3) rapid freezing 10min, 13200rpm/min on ice after water-bath, centrifugal 3min, gets supernatant, preserves, for subsequent PCR amplification.
PCR detects:
1, increase to Cauliflower EST-SSR Purity primer HYC33, primer sequence is:
HYC33-F：5'-AGACTTCGTCGATCCACCTG-3'
HYC33-R：5'-GACCTCGTCCTCCTCTTCCT-3'
2, reaction system:
2 ×
the each 0.25 μ L of Master Mixes5 μ L, HYC33 upstream and downstream primer 10 μm of ol/L, experimental cultivar genomic DNA template (100ng/ μ L) 1 μ L, distilled water supplies 10 μ L.
3, response procedures:
95 DEG C of denaturation 2min, 32 amplification cycles (94 DEG C of 40sec, 56 DEG C of 45sec, 72 DEG C of 1min); 72 DEG C extend 7min, 4 DEG C of preservations.
4, Product Identification: 8% non denatured polyacrylate hydrogel electrophoresis
Deposition condition, voltage: 250V; Time: 30min.
Silver dye is observed, and does molecular weight marker with 20bp Marker.
5, result: Cauliflower HYC33 amplification is shown in Fig. 4.Wherein, 1-5 is the maternal O5H2 amplified production that the inventive method extracts DNA; 6-25 is 20 strains " Tianjin product 70 " the F1 generation cross-fertilize seed Cauliflower amplification that the inventive method extracts DNA; 26-30 is male parent G × H amplification that the inventive method extracts DNA.
Embodiment 2
Be sample for examination Chinese cabbage cultivar " autumn green 75 " cross-fertilize seed, adopt the inventive method DNA rapid extraction.Preparation method is as follows:
(1) get cabbage hybrid individual plant seedling to be about 100mg and to add in 1.5mL sterile centrifugation tube, utilize the broken instrument of hand electric to be ground to powder;
(2) add 100 μ L10%Chelex-100 solution, vortex fully mixes, and is placed in 65 DEG C of water-bath water bath with thermostatic control 20min;
(3) rapid freezing 10min, 13200rpm/min on ice after water-bath, centrifugal 3min, gets supernatant, preserves, for subsequent PCR amplification.
PCR detects:
1, increase to Chinese cabbage EST-SSR Purity primer BC1, primer sequence is:
BC1-F：5'-TTTGTCCCTATTGCTCAGGG-3'
BC1-R：5'-CCGAGAACGTCTTCTCCTTG-3'
2, reaction system:
2 ×
the each 0.25 μ L of Master Mixes5 μ L, upstream and downstream primer 10 μm of ol/L, experimental cultivar Chinese cabbage genomic DNA template, concentration 50ng/ μ L, 1 μ L, distilled water supplies 10 μ L.
3, response procedures:
PCR response procedures is 94 DEG C of denaturation 5min, 35 amplification cycles (94 DEG C of 1min, 53.5 DEG C of 45sec, 72 DEG C of 45sec); 72 DEG C extend 7min, 4 DEG C of preservations.
4, Product Identification: 8% non denatured polyacrylate hydrogel electrophoresis
Deposition condition, voltage: 250V; Time: 30min.
Silver dye is observed, and does molecular weight marker with 20bp Marker.
5, result: Chinese cabbage BC1 amplification is shown in Fig. 5.Wherein, M is 20bpMarker; 1-43 is 43 strain Chinese cabbage cultivars " autumn green 75 " the cross-fertilize seed amplification that the inventive method extracts DNA.
Embodiment 3
Be sample for examination Chinese cabbage cultivar " Tianjin white 56 " cross-fertilize seed, adopt the inventive method DNA rapid extraction.Preparation method is as follows:
(1) get cabbage hybrid individual plant seedling to be about 100mg and to add in 1.5mL sterile centrifugation tube, utilize the broken instrument of hand electric to be ground to powder;
(2) add 100 μ L10%Chelex-100 solution, vortex fully mixes, and is placed in 65 DEG C of water-bath water bath with thermostatic control 20min;
(3) rapid freezing 10min, 13200rpm/min on ice after water-bath, centrifugal 3min, gets supernatant, preserves, for subsequent PCR amplification.
PCR detects:
1, increase to Chinese cabbage EST-SSR Purity primer BC9, primer sequence is:
BC9-F：5'-ACCTTCCGTCTCTGGGTCTT-3'
BC9-R：5'-TTTTGAACGAGGTGGAGGAC-3'
2, reaction system:
2 ×
the each 0.25 μ L of Master Mixes5 μ L, upstream and downstream primer 10 μm of ol/L, experimental cultivar Chinese cabbage genomic DNA template, concentration 50ng/ μ L, 1 μ L, distilled water supplies 10 μ L.
3, response procedures:
PCR response procedures is 94 DEG C of denaturation 5min, 35 amplification cycles (94 DEG C of 1min, 53.5 DEG C of 45sec, 72 DEG C of 45sec); 72 DEG C extend 7min, 4 DEG C of preservations.
4, Product Identification: 8% non denatured polyacrylate hydrogel electrophoresis
Deposition condition, voltage: 250V; Time: 30min.
Silver dye is observed, and does molecular weight marker with 20bp Marker.
5, result: Chinese cabbage BC9 amplification is shown in Fig. 6.Wherein, M is 20bpMarker; 1-37 is 37 strain Chinese cabbage cultivars " Tianjin white 56 " the cross-fertilize seed amplification that the inventive method extracts DNA.
Embodiment 4
Be sample for examination Chinese cabbage cultivar " autumn green 60 " cross-fertilize seed, adopt the inventive method DNA rapid extraction.Preparation method is as follows:
(1) get cabbage hybrid individual plant seedling to be about 100mg and to add in 1.5mL sterile centrifugation tube, utilize the broken instrument of hand electric to be ground to powder;
(2) add 100 μ L10%Chelex-100 solution, vortex fully mixes, and is placed in 65 DEG C of water-bath water bath with thermostatic control 20min;
(3) rapid freezing 10min, 13200rpm/min on ice after water-bath, centrifugal 3min, gets supernatant, preserves, for subsequent PCR amplification.
PCR detects:
1, increase to Chinese cabbage EST-SSR Purity primer BC9, primer sequence is:
BC26-F：5'-ATAACAACAACCTGCCCGAG-3'
BC26-R：5'-GAAGCACAAGAACAGGGCTC-3'
2, reaction system:
2 ×
the each 0.25 μ L of Master Mixes5 μ L, upstream and downstream primer 10 μm of ol/L, experimental cultivar Chinese cabbage genomic DNA template, concentration 50ng/ μ L, 1 μ L, distilled water supplies 10 μ L.
3, response procedures:
PCR response procedures is 94 DEG C of denaturation 5min, 35 amplification cycles (94 DEG C of 1min, 53.5 DEG C of 45sec, 72 DEG C of 45sec); 72 DEG C extend 7min, 4 DEG C of preservations.
4, Product Identification: 8% non denatured polyacrylate hydrogel electrophoresis
Deposition condition, voltage: 250V; Time: 30min.
Silver dye is observed, and does molecular weight marker with 20bp Marker.
5, result: Chinese cabbage BC9 amplification is shown in Fig. 7.Wherein, M is 20bpMarker; 1-46 is 46 strain Chinese cabbage cultivars " autumn green 60 " the cross-fertilize seed amplification that the inventive method extracts DNA.
Claims (4)
1. a method of high-throughput rapid extraction vegetables single seed DNA, is characterized in that it carries out as follows:
(1) get plant sample 100mg to be measured to add in 1.5mL sterile centrifugation tube, utilize the broken instrument of hand electric to be ground to powder;
(2) add 100 μ L10%Chelex-100 solution, vortex fully mixes, and is placed in 65 DEG C of water-bath water bath with thermostatic control 20min;
(3) rapid freezing 10min, 13200rpm/min on ice after water-bath, centrifugal 3min, gets supernatant, preserves, for subsequent PCR amplification.
2. extracting method as claimed in claim 1, wherein said sample to be tested is that plant sample comprises all kinds of vegetable plant samples such as Cauliflower, Chinese cabbage, cucumber, celery.
3. extracting method as claimed in claim 1, wherein said sample to be tested is the fresh individual plant sample that plant seed broken shell is developed to initial stage seedling.
4. the method for claim 1, wherein said Chelex-100 strength of solution is 10%, and add-on is 100 μ L; Water bath with thermostatic control temperature is 65 DEG C, and the time is 20min; Centrifugal speed is 13200rpm/min, and the time is 3min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410051556.3A CN104845963A (en) | 2014-02-14 | 2014-02-14 | Method for high flux rapid extraction of vegetable single seed DNA |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410051556.3A CN104845963A (en) | 2014-02-14 | 2014-02-14 | Method for high flux rapid extraction of vegetable single seed DNA |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104845963A true CN104845963A (en) | 2015-08-19 |
Family
ID=53845952
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410051556.3A Pending CN104845963A (en) | 2014-02-14 | 2014-02-14 | Method for high flux rapid extraction of vegetable single seed DNA |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104845963A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2679907C1 (en) * | 2018-03-12 | 2019-02-14 | Федеральное государственное бюджетное образовательное учреждение дополнительного профессионального образования "Российская медицинская академия непрерывного профессионального образования" Министерства здравоохранения Российской Федерации (ФГБОУ ДПО РМАНПО Минздрава России) | Method for express dna extraction from unfrozen blood |
| CN109619659A (en) * | 2018-12-03 | 2019-04-16 | 云南瑞升烟草技术（集团）有限公司 | Preparation method of tobacco nucleic acid extract and application of tobacco nucleic acid extract in tobacco |
| CN110541045A (en) * | 2019-09-16 | 2019-12-06 | 天津大学 | Method for rapidly detecting molecular characteristics and variety purity by using corn seeds |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1740338A (en) * | 2001-03-28 | 2006-03-01 | 科学与工业研究委员会 | DnA marker for estimating seeds purity and method for utilizing DNA sequence to estimate seeds purity |
| CN101220390A (en) * | 2007-12-18 | 2008-07-16 | 天津市农业科学院中心实验室 | Method for rapidly extracting plants sample DNA |
-
2014
- 2014-02-14 CN CN201410051556.3A patent/CN104845963A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1740338A (en) * | 2001-03-28 | 2006-03-01 | 科学与工业研究委员会 | DnA marker for estimating seeds purity and method for utilizing DNA sequence to estimate seeds purity |
| CN101220390A (en) * | 2007-12-18 | 2008-07-16 | 天津市农业科学院中心实验室 | Method for rapidly extracting plants sample DNA |
Non-Patent Citations (3)
| Title |
|---|
| J. CHUNWONGSE等: "Pre-germination genotypic screening using PCR amplification of half-seeds", 《THEOR APPL GENET》 * |
| KWON HWANGBO等: "Rapid and simple method for DNA extraction from plant and algal species suitable for PCR amplification using a chelating resin Chelex 100", 《PLANT BIOTECHNOL REP》 * |
| 蔡翠霞等: "改良Chelex-100法快速提取转基因农产品DNA", 《生物技术通报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2679907C1 (en) * | 2018-03-12 | 2019-02-14 | Федеральное государственное бюджетное образовательное учреждение дополнительного профессионального образования "Российская медицинская академия непрерывного профессионального образования" Министерства здравоохранения Российской Федерации (ФГБОУ ДПО РМАНПО Минздрава России) | Method for express dna extraction from unfrozen blood |
| CN109619659A (en) * | 2018-12-03 | 2019-04-16 | 云南瑞升烟草技术（集团）有限公司 | Preparation method of tobacco nucleic acid extract and application of tobacco nucleic acid extract in tobacco |
| CN109619659B (en) * | 2018-12-03 | 2022-05-13 | 云南瑞升烟草技术（集团）有限公司 | Application of tobacco nucleic acid extract in tobacco |
| CN110541045A (en) * | 2019-09-16 | 2019-12-06 | 天津大学 | Method for rapidly detecting molecular characteristics and variety purity by using corn seeds |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105349532A (en) | Method and kit for extracting free nucleic acid by using paramagnetic particle method | |
| EP4219743A1 (en) | Sample nucleic acid measurement test kit, reagent, and application thereof | |
| CN105368820A (en) | Whole blood DNA (deoxyribonucleic acid) extraction kit based on paramagnetic particle method and application of extraction kit | |
| CN105112400A (en) | Kit for extracting free DNA | |
| CN110567951B (en) | Apple stem groove virus visual detection system based on CRISPR-Cas12a technology and detection method thereof | |
| CN105420226A (en) | Animal tissue DNA extracting kit based on paramagnetic particle method and application of animal tissue DNA extracting kit | |
| CN103602738B (en) | Multiple PCR (Polymerase Chain Reaction) primer and method for quickly identifying variety of scallops | |
| WO2018006458A1 (en) | Fungal microassay method using single-cell sequencing and kit | |
| CN104962553A (en) | Using method for extracting virus DNA by using micro-nucleic acid releasing agent | |
| CN107964545A (en) | A kind of kit and its method based on magnetic bead technology extraction fungi/bacterial genomes DNA | |
| CN102146112B (en) | Method for extracting desoxyribonucleic acid from formalin fixed and paraffin embedded tissues | |
| CN108070585A (en) | A kind of kit and its method based on magnetic bead technology extraction poba gene group DNA | |
| CN105018485A (en) | Primer and probe for detecting peste des petits ruminants virus by virtue of RPA (Recombinase Polymerase Amplification) technique | |
| CN104845963A (en) | Method for high flux rapid extraction of vegetable single seed DNA | |
| CN107653333A (en) | A kind of ox Babesia nest-type PRC specific primer and detection kit and nested PCR detection method | |
| CN104611324A (en) | Whole blood sample fast pyrolysis kit and detection method thereof | |
| CN102094077A (en) | Kit for detecting genotype of mycobacterium tuberculosis clinical isolation strain quickly | |
| CN108070636A (en) | A kind of processing method and kit of fluorescent PCR amplified sample | |
| CN108866205A (en) | Identify the specific primer of hiruto based on molecular biology method | |
| CN100371461C (en) | Swine red cell body PCR detecting method | |
| CN104988240B (en) | Differentiate the method for bee colony Higher production royal jelly character using SNP marker rs16287910 | |
| CN109371110B (en) | LAMP (loop-mediated isothermal amplification) detection kit for bacterial canker pathogen of poplar | |
| CN106987587A (en) | A kind of rapid extraction nuclei aoid methods for fluorescence quantitative PCR detection | |
| CN108841991B (en) | Poplar chloroplast whole genome PCR amplification primer and application thereof | |
| CN116377118A (en) | Multiplex PCR detection method for three pathogenic bacteria of Solanaceae and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20150819 |