CN102936592A - Fast and high-efficiency extracting and purifying method of corn mitochondrial DNA (deoxyribonucleic acid) - Google Patents
Fast and high-efficiency extracting and purifying method of corn mitochondrial DNA (deoxyribonucleic acid) Download PDFInfo
- Publication number
- CN102936592A CN102936592A CN2012105193529A CN201210519352A CN102936592A CN 102936592 A CN102936592 A CN 102936592A CN 2012105193529 A CN2012105193529 A CN 2012105193529A CN 201210519352 A CN201210519352 A CN 201210519352A CN 102936592 A CN102936592 A CN 102936592A
- Authority
- CN
- China
- Prior art keywords
- centrifugal
- add
- volume
- pipe
- precipitation
- 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
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a fast and high-efficiency extracting and purifying method of corn mitochondrial DNA (deoxyribonucleic acid). The extracting and purifying method comprises the steps of charging corn explants into a pre-cooled mortar in a splitting manner, adding 4mL of pre-cooled extraction buffer solution A in 1g of sample, adding 1-2g of quartz sands, and grinding to be a homogenate; taking three 10mL centrifugal tubes, respectively numbering the centrifugal tubes as A, B and C, then taking three 2mL centrifugal tubes and respectively numbering the centrifugal tubes as D, E and F; and in the extraction process, processing with RNase A and RNase T1 simultaneously to completely remove RNA (ribonucleic acid) pollution. MgCl2 has the function of DNaseI buffer so as to be capable of replacing DNaseI buffer, and the cost is lowered. Due to the adoption of the method disclosed by the invention, the output rate of mtDNA is improved obviously.
Description
Technical field
What the present invention relates to is extraction and the purification process of a kind of rapidly and efficiently Corn Mitochondria DNA.
Background technology
Plant mtDNA extracting method mainly contains the methods such as density gradient centrifugation, differential centrifugation; Though the Mitochondrial DNA purity that density gradient centrifugation obtains is high, complicated operation, consuming time, and also large to the demand of material, the mtDNA productive rate is low simultaneously, and long-time high speed centrifugation also requires very high to whizzer; Utilize density gradient centrifugation to extract the Mitochondrial DNA experimental result as shown in Figure 1, its master tape is unintelligible, and the band disperse is seriously polluted, and loaded down with trivial details, the consuming time length of process; Although common differential centrifugation is simple to operate, low, the easy degraded of the mtDNA purity of extracting, and the contaminating impurities such as nuclear DNA, RNA and protein are serious, can not satisfy the follow-up study requirements of one's work; High salt-Proteinase K method extraction Mitochondrial DNA has simple to operate, and the mtDNA that extracts has structural integrity, purity and productive rate than advantages of higher, can satisfy the needs of pcr analysis, the assignment of genes gene mapping, the operation of clone's equimolecular.
Summary of the invention
Technical problem to be solved by this invention is extraction and the purification process that a kind of rapidly and efficiently Corn Mitochondria DNA is provided for the deficiencies in the prior art.
Technical scheme of the present invention is as follows:
Extraction and the purification process of a kind of rapidly and efficiently Corn Mitochondria DNA may further comprise the steps:
(1) the corn explant is divided in the mortar that is filled to precooling, add the Extraction buffer A of precooling in the ratio of every gram sample 4mL, put into 1-2g quartz sand, be ground to homogenate; The centrifuge tube of getting 3 10mL is numbered respectively A, B, C, and the centrifuge tube of getting again 3 2mL is numbered respectively D, E, F.
(2) filter homogenate with 6 layers of sterile gauze, will grind tissue and put back in the mortar, add buffer A by every gram sample 2mL and again grind and filter, 2 times filtrate merges.To install to filtrate dividing in the A pipe, the centrifugal 10min of 500r/min removes large stretch of fragment tissue;
(3) supernatant liquor is transferred in the B pipe, centrifugal 2 times of 2600r/min is respectively 15 and 10min, removes the plastids such as chloroplast(id);
(4) get supernatant liquor to the C pipe, centrifugal 15 min of 10000 r/min abandon supernatant, add the A liquid of 1 mL precooling in precipitation, blow afloat gently precipitation, mixing with the rifle head.The centrifugal 10min of 1000r/min sucks supernatant in the D pipe, adds the 100mg/mL DNaseI of 1/500 times of volume and the 1mol/L MgCl of 1/100 times of volume
2The 0.5mol/L Na that adds 1/25 times of volume behind the ice bath 1h
2EDTA leaves standstill 10 min, stops the DNaseI reaction;
(5) the centrifugal 15min of 13200r/min removes supernatant, obtains the plastosome precipitation, adds ST liquid purifying 1 time again, obtains the plastosome of purifying;
(6) add the 1mL lysate B precipitation that suspends in the D pipe precipitation, add again 25mg/mL Proteinase K 2.5 μ L, 50 ℃ of incubation 1 h, 37 ℃ of incubation 1h again, during the interval shake;
(7) take out the centrifuge tube room temperature and place, after its cooling, add isopyknic phenol: chloroform: the primary isoamyl alcohol mixed solution, mixed solution mixed volume ratio is 25:24:1, fully mixing is placed on and shakes 15min on the shaking table, centrifugal 10 min of 10000 r/min;
(8) turn supernatant liquor to the E pipe, add isopyknic chloroform: the primary isoamyl alcohol mixed solution, mixed solution mixed volume ratio is 24:1, turns upside down to shake up approximately 10min centrifugal 10 min of 10000 r/min;
(9) supernatant liquor is transferred in the F pipe, then adds the RNaseT of the 1000U/ μ L of the RNaseA of 1/100 volume 10mg/mL and 1/500 volume
1, RNA is removed in 37 ℃ of water-baths 1 hour;
(10) add isopyknic chloroform: the primary isoamyl alcohol mixed solution, mixed solution mixed volume ratio 24:1, turning upside down shakes up approximately 10min, centrifugal 10 min of 10000 r/min;
(11) get 0. 1 times of volume NaAc and 2 times of volume dehydrated alcohols that supernatant liquor adds precooling to the centrifuge tube of 1.5mL ,-20 ℃ are spent the night;
(12) 4 ℃, the centrifugal 15min of 13200r/min abandon supernatant, and precipitation is with 75% washing with alcohol 2 ~ 3 times;
(13) dissolve in a small amount of water after being deposited in air drying ,-20 ℃ of refrigerations are for subsequent use.
Used reagent wherein:
(1) buffer A: 1mol/L Tris-HCl 12.5mL, 0.5mol/L Na
2EDTA 12.5mL, 1.3 mol/L NaCl 15.743g are settled to 250mL with the sterilization distilled water, autoclave sterilization, room temperature preservation.Add 0.5g BSA, 0.125g halfcystine, 750 μ L beta-mercaptoethanols before using.
(2) ST liquid: sucrose 13.6916g, 1mol/L Tris-HCl 5mL, 0.5mol/L Na
2EDTA 5mL is settled to 100mL with the sterilization distilled water, autoclave sterilization, room temperature preservation.Add 0.1g BSA before using.
(3) lysate B:1mol/L Tris-HCl 2.5mL, 0.5mol/L Na
2EDTA 4mL, 10% SDS 5mL is settled to 100mL with the sterilization distilled water, room temperature preservation.
Beneficial effect of the present invention:
(1) this experiment is adopted two kinds of RNA enzyme RNase A and RNase T in leaching process
1Process simultaneously the pollution that can remove RNA fully.
(2) MgCl
2Effect with DNaseI buffer, alternative DNaseI buffer reduces cost.
(3) output capacity of mtDNA is improved significantly.
Description of drawings
Fig. 1 is for utilizing density gradient centrifugation to extract the Mitochondrial DNA experimental result;
Fig. 2 is for adopting method of the present invention to extract the Mitochondrial DNA experimental result;
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.
(1) the corn explant is divided in the mortar that is filled to precooling, add the Extraction buffer A of precooling in the ratio of every gram sample 4mL, put into 1-2g quartz sand, be ground to homogenate; The centrifuge tube of getting 3 10mL is numbered respectively A, B, C, and the centrifuge tube of getting again 3 2mL is numbered respectively D, E, F.
(2) filter homogenate with 6 layers of sterile gauze, will grind tissue and put back in the mortar, add buffer A by every gram sample 2mL and again grind and filter, 2 times filtrate merges.To install to filtrate dividing in the A pipe, the centrifugal 10min of 500r/min removes large stretch of fragment tissue;
(3) supernatant liquor is transferred in the B pipe, centrifugal 2 times of 2600r/min is respectively 15 and 10min, removes the plastids such as chloroplast(id);
(4) get supernatant liquor to the C pipe, centrifugal 15 min of 10000 r/min abandon supernatant, add the A liquid of 1 mL precooling in precipitation, blow afloat gently precipitation, mixing with the rifle head.The centrifugal 10min of 1000r/min sucks supernatant in the D pipe, adds the 100mg/mL DNaseI of 1/500 times of volume and the 1mol/L MgCl of 1/100 times of volume
2The 0.5mol/L Na that adds 1/25 times of volume behind the ice bath 1h
2EDTA leaves standstill 10 min, stops the DNaseI reaction;
(5) the centrifugal 15min of 13200r/min removes supernatant, obtains the plastosome precipitation, adds ST liquid purifying 1 time again, obtains the plastosome of purifying;
(6) add the 1mL lysate B precipitation that suspends in the D pipe precipitation, add again 25mg/mL Proteinase K 2.5 μ L, 50 ℃ of incubation 1 h, 37 ℃ of incubation 1h again, during the interval shake;
(7) take out the centrifuge tube room temperature and place, after its cooling, add isopyknic phenol: chloroform: the primary isoamyl alcohol mixed solution, mixed solution mixed volume ratio is 25:24:1, fully mixing is placed on and shakes 15min on the shaking table, centrifugal 10 min of 10000 r/min;
(8) turn supernatant liquor to the E pipe, add isopyknic chloroform: the primary isoamyl alcohol mixed solution, mixed solution mixed volume ratio is 24:1, turns upside down to shake up approximately 10min centrifugal 10 min of 10000 r/min;
(9) supernatant liquor is transferred in the F pipe, then adds the RNaseT of the 1000U/ μ L of the RNaseA of 1/100 volume 10mg/mL and 1/500 volume
1RNA is removed in 37 ℃ of water-baths 1 hour;
(10) add isopyknic chloroform: the primary isoamyl alcohol mixed solution, mixed solution mixed volume ratio 24:1, turning upside down shakes up approximately 10min, centrifugal 10 min of 10000 r/min;
(11) get 0. 1 times of volume NaAc and 2 times of volume dehydrated alcohols that supernatant liquor adds precooling to the centrifuge tube of 1.5mL ,-20 ℃ are spent the night;
(12) 4 ℃, the centrifugal 15min of 13200r/min abandon supernatant, and precipitation is with 75% washing with alcohol 2 ~ 3 times;
(13) dissolve in a small amount of water after being deposited in air drying ,-20 ℃ of refrigerations are for subsequent use.
Reagent
(1) buffer A: 1mol/L Tris-HCl 12.5mL, 0.5mol/L Na
2EDTA 12.5mL, 1.3 mol/L NaCl 15.743g are settled to 250mL with the sterilization distilled water, autoclave sterilization, room temperature preservation.Add 0.5g BSA, 0.125g halfcystine, 750 μ L beta-mercaptoethanols before using.
(2) ST liquid: sucrose 13.6916g, 1mol/L Tris-HCl 5mL, 0.5mol/L Na
2EDTA 5mL is settled to 100mL with the sterilization distilled water, autoclave sterilization, room temperature preservation.Add 0.1g BSA before using.
(3) lysate B:1mol/L Tris-HCl 2.5mL, 0.5mol/L Na
2EDTA 4mL, 10% SDS 5mL is settled to 100mL with the sterilization distilled water, room temperature preservation.
As shown in Figure 2, can find out near the point sample hole have RNA to pollute without macromole DNA pollution and front end.Measure its OD with the nucleic acid-protein instrument
260/ OD
280And concentration, the result shows OD
260/ OD
280All between 1.8-2.0, show that mtDNA purity is higher, protein contamination is less.
Should be understood that, for those of ordinary skills, can be improved according to the above description or conversion, and all these improvement and conversion all should belong to the protection domain of claims of the present invention.
Claims (1)
1. rapidly and efficiently extraction and the purification process of Corn Mitochondria DNA is characterized in that, may further comprise the steps:
(1) the corn explant is divided in the mortar that is filled to precooling, add the Extraction buffer A of precooling in the ratio of every gram sample 4mL, put into 1-2g quartz sand, be ground to homogenate; The centrifuge tube of getting 3 10mL is numbered respectively A, B, C, and the centrifuge tube of getting again 3 2mL is numbered respectively D, E, F;
(2) filter homogenate with 6 layers of sterile gauze, will grind tissue and put back in the mortar, add buffer A by every gram sample 2mL and again grind and filter, 2 times filtrate merges; To install to filtrate dividing in the A pipe, the centrifugal 10min of 500r/min removes large stretch of fragment tissue;
(3) supernatant liquor is transferred in the B pipe, centrifugal 2 times of 2600r/min is respectively 15 and 10min, removes the plastids such as chloroplast(id);
(4) get supernatant liquor to the C pipe, centrifugal 15 min of 10000 r/min abandon supernatant, add the A liquid of 1 mL precooling in precipitation, blow afloat gently precipitation, mixing with the rifle head; The centrifugal 10min of 1000r/min sucks supernatant in the D pipe, adds the 100mg/mL DNaseI of 1/500 times of volume and the 1mol/L MgCl of 1/100 times of volume
2The 0.5mol/L Na that adds 1/25 times of volume behind the ice bath 1h
2EDTA leaves standstill 10 min, stops the DNaseI reaction;
(5) the centrifugal 15min of 13200r/min removes supernatant, obtains the plastosome precipitation, adds ST liquid purifying 1 time again, obtains the plastosome of purifying;
(6) add the 1mL lysate B precipitation that suspends in the D pipe precipitation, add again 25mg/mL Proteinase K 2.5 μ L, 50 ℃ of incubation 1 h, 37 ℃ of incubation 1h again, during the interval shake;
(7) take out the centrifuge tube room temperature and place, after its cooling, add isopyknic phenol: chloroform: the primary isoamyl alcohol mixed solution, mixed solution mixed volume ratio is 25:24:1, fully mixing is placed on and shakes 15min on the shaking table, centrifugal 10 min of 10000 r/min;
(8) turn supernatant liquor to the E pipe, add isopyknic chloroform: the primary isoamyl alcohol mixed solution, mixed solution mixed volume ratio is 24:1, turns upside down to shake up approximately 10min centrifugal 10 min of 10000 r/min;
(9) supernatant liquor is transferred in the F pipe, then adds the RNaseT of the 1000U/ μ L of the RNaseA of 1/100 volume 10mg/mL and 1/500 volume
1RNA is removed in 37 ℃ of water-baths 1 hour;
(10) add isopyknic chloroform: the primary isoamyl alcohol mixed solution, mixed solution mixed volume ratio 24:1, turning upside down shakes up approximately 10min, centrifugal 10 min of 10000 r/min;
(11) get 0. 1 times of volume NaAc and 2 times of volume dehydrated alcohols that supernatant liquor adds precooling to the centrifuge tube of 1.5mL ,-20 ℃ are spent the night;
(12) 4 ℃, the centrifugal 15min of 13200r/min abandon supernatant, and precipitation is with 75% washing with alcohol 2 ~ 3 times;
(13) dissolve in a small amount of water after being deposited in air drying ,-20 ℃ of refrigerations are for subsequent use;
Used reagent wherein:
(1) buffer A: 1mol/L Tris-HCl 12.5mL, 0.5mol/L Na
2EDTA 12.5mL, 1.3 mol/L NaCl 15.743g are settled to 250mL with the sterilization distilled water, autoclave sterilization, room temperature preservation; Add 0.5g BSA, 0.125g halfcystine, 750 μ L beta-mercaptoethanols before using;
(2) ST liquid: sucrose 13.6916g, 1mol/L Tris-HCl 5mL, 0.5mol/L Na
2EDTA 5mL is settled to 100mL with the sterilization distilled water, autoclave sterilization, room temperature preservation; Add 0.1g BSA before using;
(3) lysate B:1mol/L Tris-HCl 2.5mL, 0.5mol/L Na
2EDTA 4mL, 10% SDS 5mL is settled to 100mL with the sterilization distilled water, room temperature preservation.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN2012105193529A CN102936592A (en) | 2012-12-06 | 2012-12-06 | Fast and high-efficiency extracting and purifying method of corn mitochondrial DNA (deoxyribonucleic acid) |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN2012105193529A CN102936592A (en) | 2012-12-06 | 2012-12-06 | Fast and high-efficiency extracting and purifying method of corn mitochondrial DNA (deoxyribonucleic acid) |
Publications (1)
Publication Number | Publication Date |
---|---|
CN102936592A true CN102936592A (en) | 2013-02-20 |
Family
ID=47695514
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN2012105193529A Pending CN102936592A (en) | 2012-12-06 | 2012-12-06 | Fast and high-efficiency extracting and purifying method of corn mitochondrial DNA (deoxyribonucleic acid) |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN102936592A (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106947729A (en) * | 2017-03-17 | 2017-07-14 | 中国农业科学院油料作物研究所 | A kind of separated in synchronization high-purity vegetable chloroplaset and mitochondrial method |
CN109077199A (en) * | 2017-06-14 | 2018-12-25 | 发贵科技(贵州)有限公司 | Vegetables mitochondria oral emulsion and preparation method thereof |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101111597A (en) * | 2005-03-04 | 2008-01-23 | 富士胶片株式会社 | Method for separating and purifying nucleic acid |
CN101899432A (en) * | 2010-05-04 | 2010-12-01 | 中国农业科学院油料作物研究所 | Method for extracting high-purity sesame mitochondrion DNA |
-
2012
- 2012-12-06 CN CN2012105193529A patent/CN102936592A/en active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101111597A (en) * | 2005-03-04 | 2008-01-23 | 富士胶片株式会社 | Method for separating and purifying nucleic acid |
CN101899432A (en) * | 2010-05-04 | 2010-12-01 | 中国农业科学院油料作物研究所 | Method for extracting high-purity sesame mitochondrion DNA |
Non-Patent Citations (4)
Title |
---|
LING HUANG ET AL.: "Expression characterization of genes for CMS-C in maize", 《PROTOPLASMA》 * |
李振兴 等: "一种优化的大白菜线粒体DNA提取方法", 《华农学报》 * |
李文强 等: "小麦线粒体DNA的高效提取方法", 《遗传》 * |
谭才邓: "植物线粒体提取的一种简捷方法", 《广东轻工职业技术学院学报》 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106947729A (en) * | 2017-03-17 | 2017-07-14 | 中国农业科学院油料作物研究所 | A kind of separated in synchronization high-purity vegetable chloroplaset and mitochondrial method |
CN106947729B (en) * | 2017-03-17 | 2019-10-08 | 中国农业科学院油料作物研究所 | A kind of method of separated in synchronization high-purity vegetable chloroplaset and mitochondria |
CN109077199A (en) * | 2017-06-14 | 2018-12-25 | 发贵科技(贵州)有限公司 | Vegetables mitochondria oral emulsion and preparation method thereof |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN104178480B (en) | Using the kit and method of DNA adsorption column rapid extraction DNA of plants | |
CN101712953B (en) | DNA extracting method for evaluating community diversity of the intestinal microorganisms of animals | |
CN102286460B (en) | Extraction of DNA in Pleurotus eryngii by improved CTAB (cetyltrimethylammonium bromide) method | |
CN105385682B (en) | The simple and easy method of rapid extraction human faecal mass DNA of bacteria | |
HRP20201452T1 (en) | Isolation of nucleic acids | |
CN103215251A (en) | Method for separating chloroplast DNA | |
CN103320425B (en) | Rapid extraction method of shellfish deoxyribonucleic acid (DNA) for large-scale and high-throughput genotyping | |
CN102936592A (en) | Fast and high-efficiency extracting and purifying method of corn mitochondrial DNA (deoxyribonucleic acid) | |
CN102660537A (en) | Method for extracting a large number of high-purity deoxyribonucleic acid (DNA) from chicken blood | |
CN104152436B (en) | DNA isolation and purification methods and its kit | |
CN102031252B (en) | Method for rapidly extracting total DNA from soil | |
CN104560953A (en) | Kit for rapidly extracting sludge microbial genome DNA and extracting method | |
CN103820434A (en) | Method for extracting total DNA (deoxyribonucleic acid) of fungal hyphae | |
CN104212795A (en) | Method for extracting total ribonucleic acid (RNA) of fish brain tissue | |
CN106834535A (en) | Specific primer, kit and discrimination method for identifying Bungarus Parvus | |
CN102703430B (en) | Method for extracting microorganism total DNA (Deoxyribonucleic Acid) in pu'er tea piling fermentation process | |
CN103882006A (en) | Preparation method and extraction method for DNA extraction reagent suitable for various samples | |
CN104694530A (en) | Extraction method of wheat genome DNA | |
CN111019940A (en) | Extracting solution for directly extracting whole blood genome DNA and extracting method thereof | |
CN111467486A (en) | Method for purifying rabies vaccine | |
CN103243088A (en) | Method for extracting high-quality DNA (deoxyribonucleic acid) in dry leaves of soybeans suitable for transgenic detection | |
CN107151666A (en) | The extracting method of microbial DNA in a kind of water body | |
CN108866042B (en) | Extraction method of trace RNA | |
CN100395257C (en) | Acid potassium ion aqua and DNA extracting method and kit therewith | |
CN101709299A (en) | Method for purifying nucleic acid by applying magnetic silica microballoon modified with cation on surface in biological sample |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C12 | Rejection of a patent application after its publication | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20130220 |