CN108913686A - A kind of pcr template DNA sample extracting method - Google Patents
A kind of pcr template DNA sample extracting method Download PDFInfo
- Publication number
- CN108913686A CN108913686A CN201810856883.4A CN201810856883A CN108913686A CN 108913686 A CN108913686 A CN 108913686A CN 201810856883 A CN201810856883 A CN 201810856883A CN 108913686 A CN108913686 A CN 108913686A
- Authority
- CN
- China
- Prior art keywords
- dna
- sodium hydroxide
- hydroxide solution
- blade
- extracting method
- 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
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
Landscapes
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Crystallography & Structural Chemistry (AREA)
- Plant Pathology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Extraction Or Liquid Replacement (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
A kind of pcr template DNA sample extracting method, includes the following steps:Fresh and tender blade grind into powder, the sodium hydroxide solution of 0.15-0.3MOL/L molar concentration is added in blade container, and solution quality is 3-10 times of leaf quality, the EDTA-2 sodium containing 5-10% mass concentration in the sodium hydroxide solution;Mixture 30-120 seconds for heating sodium hydroxide solution and blade powder with boiling water;Centrifugation is layered suspension after shaking up, and takes supernatant, and supernatant is diluted 5-20 times with the Tris-HCl that 1-10MOL/ rises, that is, obtains DNA of plants sample.Using pcr template of the present invention DNA sample extracting method, DNA can be quickly and easily extracted, and reduces the oxidative phenomena in DNA of plants extraction process, improves DNA recovery rate.
Description
Technical field
The invention belongs to field of biotechnology, are related to DNA of plants sample extraction, and in particular to a kind of pcr template DNA sample
This extracting method.
Background technique
DNA, that is, DNA is the organic compound of molecular structure complexity.As chromosome an ingredient and deposit
It is in nucleus.Function is storage hereditary information.DNA molecule is huge, is made of nucleotide.The extraction of DNA of plants is plant
A variety of methods such as molecular biology and a kind of basic technology, including CTAB, SDS of genetic engineering research；DNA activity is high,
It is oxidized easily;In the prior art in plant extract DNA, DNA, which is oxidized easily, causes the rotten damage of picodna sample.
PCR (polymerase chain reaction）It is that 95 ° of high temperature time variations Celsius will become single-stranded, low temperature in vitro using DNA（Through
It is often 60 °C or so）When the primer and single-stranded principle by base pair complementarity, denaturation unwinding can also occur at high temperature for DNA,
Double-strand can be become again after temperature reduces with renaturation.Therefore, the denaturation and renaturation of DNA are controlled by temperature change, and design is added
Primer, archaeal dna polymerase, dNTP can complete the replication in vitro of specific gene.But archaeal dna polymerase can inactivate at high temperature, because
This, new archaeal dna polymerase must be all added in circulation every time, not only operate loaded down with trivial details but also expensive, constrain answering for round pcr
With and development.
Summary of the invention
To overcome technological deficiency of the existing technology, the invention discloses a kind of pcr template DNA sample extraction sides
Method.
Pcr template of the present invention DNA sample extracting method, includes the following steps:
The sodium hydroxide solution of 0.15-0.3MOL/L molar concentration, solution are added in blade container for fresh and tender blade grind into powder
Quality is 3-10 times of leaf quality, the EDTA-2 sodium containing 5-10% mass concentration in the sodium hydroxide solution;
Mixture 30-120 seconds for heating sodium hydroxide solution and blade powder with boiling water;Centrifugation is layered suspension after shaking up,
Supernatant is taken, supernatant is diluted 5-20 times with the Tris-HCl that 1-10MOL/ rises, that is, obtains DNA of plants sample.
Preferably, the fresh and tender blade grind into powder is carried out under immersing in liquid nitrogen environment.
Using pcr template of the present invention DNA sample extracting method, DNA can be quickly and easily extracted, and reduces plant
Oxidative phenomena in object DNA extraction process improves DNA recovery rate.
Specific embodiment
Specific embodiments of the present invention will be described in further detail below.
Pcr template of the present invention DNA sample extracting method, includes the following steps:
The sodium hydroxide solution of 0.15-0.3MOL/L molar concentration, solution are added in blade container for fresh and tender blade grind into powder
Quality is 3-10 times of leaf quality, the EDTA-2 sodium containing 5-10% mass concentration in the sodium hydroxide solution;
Mixture 30-120 seconds for heating sodium hydroxide solution and blade powder with boiling water;Centrifugation is layered suspension after shaking up,
Supernatant is taken, supernatant is diluted 5-20 times with the Tris-HCl that 1-10MOL/ rises, that is, obtains DNA of plants sample.
DNA of plants, which extracts sample, should select young leaflet tablet, such as select old leaf, contain a large amount of polysaccharide in old leaf and phenols is miscellaneous
Matter is unfavorable for DNA extraction.
Process of lapping can carry out under immersing in liquid nitrogen environment, avoid the air oxidation in process of lapping, liquid nitrogen cryogenics
Conducive to grinding bring heat is taken away, DNA heat damage is avoided.
After the completion of grinding, alkaline solution of sodium hydroxide is poured into, the effect of sodium hydroxide is lytic cell wall；For anti-block
Change, EDTA-2 sodium also known as disodium ethylene diamine tetraacetate should be added in sodium hydroxide solution, EDTA-2 sodium has during broken wall
Anti-oxidation effect, and the metal ion in water can be solidified and form complex compound, while there is faintly acid, it is anti-to can be used as buffer control
It answers speed, reduces reaction fever, EDTA-2 sodium can volatilize rapidly after the heating, remain in the solution minimum, avoid influencing subsequent
Reaction.
Sodium hydroxide after the reaction was completed, is heated with boiling water bath, and General reactions are completed in a test tube, and test tube is impregnated
In boiling water, boiling water bath makes reaction temperature in 90-100 degree Celsius range, and the temperature range is most useful for cracking nucleic acid.
After the completion of cracking, shaking up centrifugation is layered suspension, extracts supernatant and is diluted with Tris-HCl, Tris-HCl is
Three（Methylol）Aminomethane functions as buffer and provides buffer environment, prevents the DNA nucleic acid extracted in supernatant from being broken
It is bad.
Specific embodiment 1
It takes 10 milligrams of fresh and tender blade grind into powder and is put into test tube, the sodium hydroxide of 0.2MOL/L molar concentration is added in test tube
100 microlitres of solution, the EDTA-2 sodium containing 5% mass concentration in the sodium hydroxide solution;
Mixture 90 seconds for heating sodium hydroxide solution and blade powder with boiling water;Centrifugation is layered suspension after shaking up, and takes
Supernatant is diluted 10 times with the Tris-HCl that 1MOL/ rises, that is, obtains DNA of plants sample by clear liquid.
Specific embodiment 2
It takes 10 milligrams of fresh and tender blade grind into powder and is put into test tube, the sodium hydroxide of 0.3MOL/L molar concentration is added in test tube
50 microlitres of solution, the EDTA-2 sodium containing 10% mass concentration in the sodium hydroxide solution;
Mixture 30 seconds for heating sodium hydroxide solution and blade powder with boiling water;Centrifugation is layered suspension after shaking up, and takes
Supernatant is diluted 10 times with the Tris-HCl that 1MOL/ rises, that is, obtains DNA of plants sample by clear liquid.
Specific embodiment 3
Take 10 milligrams of fresh and tender blades, grind into powder and be put into test tube under immersing in liquid nitrogen, be added in test tube 0.3MOL/L moles it is dense
100 microlitres of the sodium hydroxide solution of degree, the EDTA-2 sodium containing 10% mass concentration in the sodium hydroxide solution;
Mixture 120 seconds for heating sodium hydroxide solution and blade powder with boiling water;Centrifugation is layered suspension after shaking up, and takes
Supernatant is diluted 3 times with the Tris-HCl that 10MOL/ rises, that is, obtains DNA of plants sample by supernatant.
Using pcr template of the present invention DNA sample extracting method, DNA can be quickly and easily extracted, and reduces plant
Oxidative phenomena in object DNA extraction process improves DNA recovery rate.
Previously described is each preferred embodiment of the invention, if the preferred embodiment in each preferred embodiment
It is not obvious contradictory or premised on a certain preferred embodiment, each preferred embodiment can any stack combinations
Use, the design parameter in the embodiment and embodiment only for the purpose of clearly stating the inventor's invention verification process, and
It is non-to limit scope of patent protection of the invention, scope of patent protection of the invention is still subject to the claims, all
It is that similarly should be included within the scope of the present invention with the variation of equivalent structure made by description of the invention.
Claims (2)
1. a kind of pcr template DNA sample extracting method, it is characterised in that, include the following steps:
The sodium hydroxide solution of 0.15-0.3MOL/L molar concentration, solution are added in blade container for fresh and tender blade grind into powder
Quality is 3-10 times of leaf quality, the EDTA-2 sodium containing 5-10% mass concentration in the sodium hydroxide solution;
Mixture 30-120 seconds for heating sodium hydroxide solution and blade powder with boiling water;Centrifugation is layered suspension after shaking up,
Supernatant is taken, supernatant is diluted 5-20 times with the Tris-HCl that 1-10MOL/ rises, that is, obtains DNA of plants sample.
2. pcr template as described in claim 1 DNA sample extracting method, which is characterized in that the fresh and tender blade is ground into
Powder is carried out under immersing in liquid nitrogen environment.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810856883.4A CN108913686A (en) | 2018-07-31 | 2018-07-31 | A kind of pcr template DNA sample extracting method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810856883.4A CN108913686A (en) | 2018-07-31 | 2018-07-31 | A kind of pcr template DNA sample extracting method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108913686A true CN108913686A (en) | 2018-11-30 |
Family
ID=64393392
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810856883.4A Pending CN108913686A (en) | 2018-07-31 | 2018-07-31 | A kind of pcr template DNA sample extracting method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108913686A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113322313A (en) * | 2021-06-19 | 2021-08-31 | 长沙理工大学 | Method for rapidly identifying plant genes |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009111336A2 (en) * | 2008-02-29 | 2009-09-11 | Shizhong Chen | Methods of purifying plasmid dna |
| CN101967473A (en) * | 2010-10-15 | 2011-02-09 | 中国水产科学研究院黑龙江水产研究所 | Method for extracting fish genome DNA |
| CN105462962A (en) * | 2016-01-15 | 2016-04-06 | 中央民族大学 | Extraction method of crocus sativus medicinal material DNA |
| CN106754879A (en) * | 2016-12-22 | 2017-05-31 | 东北农业大学 | A kind of plant leaf blade DNA simplifies rapid extracting method |
-
2018
- 2018-07-31 CN CN201810856883.4A patent/CN108913686A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009111336A2 (en) * | 2008-02-29 | 2009-09-11 | Shizhong Chen | Methods of purifying plasmid dna |
| CN101967473A (en) * | 2010-10-15 | 2011-02-09 | 中国水产科学研究院黑龙江水产研究所 | Method for extracting fish genome DNA |
| CN105462962A (en) * | 2016-01-15 | 2016-04-06 | 中央民族大学 | Extraction method of crocus sativus medicinal material DNA |
| CN106754879A (en) * | 2016-12-22 | 2017-05-31 | 东北农业大学 | A kind of plant leaf blade DNA simplifies rapid extracting method |
Non-Patent Citations (3)
| Title |
|---|
| PACKEISER H等: "An extremely simple and effective colony PCR procedure for bacteria,yeasts, and microalgae", 《APPL BIOCHEM BIOTECHNOL》 * |
| 于东明等: "煮沸裂解法快速提取Tg2576小鼠DNA", 《中国实用神经疾病杂志》 * |
| 刘阳等: "大花蕙兰子房基因组DNA提取方法的比较研究 ", 《天津农学院学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113322313A (en) * | 2021-06-19 | 2021-08-31 | 长沙理工大学 | Method for rapidly identifying plant genes |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105112445B (en) | A kind of miR-205 gene knockout kits based on CRISPR-Cas9 gene Knockouts | |
| Ortmann et al. | Transcriptome analysis of infection of the archaeon Sulfolobus solfataricus with Sulfolobus turreted icosahedral virus | |
| JP2010502197A5 (en) | ||
| CN104818253B (en) | Target transformation of site-directed integration Chinese hamster ovary celI system and application thereof | |
| CN106916848A (en) | A kind of method that gene transient expression is realized in Peach fruits | |
| CN104178480B (en) | Using the kit and method of DNA adsorption column rapid extraction DNA of plants | |
| CN105861414B (en) | A kind of preparation method of alfalfa protoplast | |
| Van Eck et al. | Agrobacterium tumefaciens-mediated transformation of Setaria viridis | |
| CN107227298A (en) | A kind of clone embryos treatment fluid and its application method and the purposes of the treatment fluid | |
| CN108913686A (en) | A kind of pcr template DNA sample extracting method | |
| CN111295443A (en) | Transposase-based genomic analysis | |
| CN105296534A (en) | Method of establishing Lycium Ruthenicum genetic transformation system and application of method | |
| Joldersma et al. | Plant genetics enters the nano age? | |
| CN104694530A (en) | Extraction method of wheat genome DNA | |
| CN107400663B (en) | Scale-purple back enteromorpha-xanthone six-position hydroxyl oxygen methyltransferase as well as coding gene and application thereof | |
| Zemska et al. | Agrobacterium-mediated insertional mutagenesis in Histoplasma capsulatum | |
| CA3152875A1 (en) | Genetically modified plants and methods of making the same | |
| CN107287227A (en) | A kind of goat miR 27a pointed decorations systems and its application | |
| CN108603160A (en) | The manufacturing method of mutant filamentous bacterium | |
| CN113897349A (en) | In-vitro large-fragment DNA cloning method based on CRISPR/Cas12a and application thereof | |
| CN110241189A (en) | A kind of long paired end sequencing method in DNA long fragment library | |
| Powers et al. | Performing ribosome profiling to assess translation in vegetative and meiotic yeast cells | |
| CN104513830B (en) | A kind of expression vector and its application suitable for Gluconobacter oxvdans | |
| CN107805641B (en) | Tobacco14-3-3cPlant expression vector of gene and application thereof | |
| CN105255801B (en) | A kind of bacterium solution and method improving lichee wilted-nut rate |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20181130 |
|
| RJ01 | Rejection of invention patent application after publication |