CN111996187A - Method for rapidly extracting buckwheat leaf genome DNA - Google Patents
Method for rapidly extracting buckwheat leaf genome DNA Download PDFInfo
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- CN111996187A CN111996187A CN202010830007.1A CN202010830007A CN111996187A CN 111996187 A CN111996187 A CN 111996187A CN 202010830007 A CN202010830007 A CN 202010830007A CN 111996187 A CN111996187 A CN 111996187A
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- 241000219051 Fagopyrum Species 0.000 title claims abstract description 39
- 235000009419 Fagopyrum esculentum Nutrition 0.000 title claims abstract description 39
- 238000000034 method Methods 0.000 title claims abstract description 26
- 239000006260 foam Substances 0.000 claims abstract description 19
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 16
- 239000000243 solution Substances 0.000 claims abstract description 15
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 12
- 238000002156 mixing Methods 0.000 claims abstract description 12
- 239000006228 supernatant Substances 0.000 claims abstract description 12
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims abstract description 8
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims abstract description 8
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims abstract description 8
- 239000011780 sodium chloride Substances 0.000 claims abstract description 8
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000007788 liquid Substances 0.000 claims abstract description 6
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 6
- 239000007983 Tris buffer Substances 0.000 claims abstract description 4
- 239000011324 bead Substances 0.000 claims abstract description 4
- 238000004140 cleaning Methods 0.000 claims abstract description 4
- 239000011521 glass Substances 0.000 claims abstract description 4
- 239000011259 mixed solution Substances 0.000 claims abstract description 4
- 239000000843 powder Substances 0.000 claims abstract description 4
- YTRQFSDWAXHJCC-UHFFFAOYSA-N chloroform;phenol Chemical compound ClC(Cl)Cl.OC1=CC=CC=C1 YTRQFSDWAXHJCC-UHFFFAOYSA-N 0.000 claims abstract description 3
- 238000001816 cooling Methods 0.000 claims abstract description 3
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 claims abstract description 3
- 238000007789 sealing Methods 0.000 claims abstract description 3
- 239000000047 product Substances 0.000 abstract 1
- 238000000605 extraction Methods 0.000 description 7
- 229960001484 edetic acid Drugs 0.000 description 4
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 3
- 230000009418 agronomic effect Effects 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- ZYWFEOZQIUMEGL-UHFFFAOYSA-N chloroform;3-methylbutan-1-ol;phenol Chemical compound ClC(Cl)Cl.CC(C)CCO.OC1=CC=CC=C1 ZYWFEOZQIUMEGL-UHFFFAOYSA-N 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000009456 molecular mechanism Effects 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 241000219050 Polygonaceae Species 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000009412 basement excavation Methods 0.000 description 1
- 238000000861 blow drying Methods 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000003631 expected effect Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000003517 fume Substances 0.000 description 1
- 239000003147 molecular marker Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000009331 sowing Methods 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
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Abstract
The invention discloses a method for rapidly extracting buckwheat leaf genome DNA, which comprises the following steps: putting buckwheat leaves and glass beads into a centrifugal tube, and putting the centrifugal tube into a foam box; pouring liquid nitrogen into the foam box, covering all centrifuge tubes, sealing the foam box and then shaking to enable internal blades to be powdered; adding an extracting solution into a centrifugal tube, completely mixing the extracting solution and the leaf powder, carrying out warm bath, adding phenol chloroform isoamylol with the same volume, uniformly mixing, and centrifuging, wherein the extracting solution comprises the following components: ethylenediaminetetraacetic acid (EDTA), Sodium Dodecyl Sulfate (SDS), sodium chloride (NaCl), and Tris (hydroxymethyl) aminomethane (Tris); centrifuging, collecting supernatant, adding equal volume of precooled isopropanol, mixing, and cooling; centrifuging the mixed solution, and removing the supernatant; adding ethanol for cleaning, centrifuging, and removing supernatant; add ddH to centrifuge tube2And dissolving O to obtain the product. The method has the advantages of short time, low cost and high efficiency.
Description
Technical Field
The invention relates to a method for extracting DNA, in particular to a method for quickly extracting buckwheat leaf genome DNA.
Background
Buckwheat is a dicotyledonous plant of buckwheat of Polygonaceae, has been planted for 2700 years in China, and has the characteristics of drought resistance, barren resistance, wide time range of sowing period, short growth period, homology of medicine and food and the like as a small crop. At present, buckwheat breeding work is carried out internationally by using modern biotechnology such as genomics. Buckwheat genomics mainly relates to important agronomic character excavation and molecular mechanism research of buckwheat, such as high yield, stress resistance, disease and insect resistance and the like. For the research of buckwheat molecular mechanism and gene function, the rapid and high-quality extraction of DNA is an important basis and precondition. However, no method for specially researching the genome DNA of the buckwheat leaves exists at present, so that the method for quickly and simply extracting the DNA of the buckwheat leaves by specially researching the buckwheat has important significance.
At present, the main extraction method of buckwheat DNA still refers to the traditional CTAB method, and the method can extract DNA samples with higher purity, but the steps are complicated, the required time is longer, generally more than 2 hours is required, the cost is higher, and the method is not suitable for high-throughput research. Therefore, the development of a simple and convenient method capable of rapidly extracting the buckwheat leaf genome DNA is important for researching the gene of the important agronomic character of buckwheat and the function thereof.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to provide a method for rapidly extracting buckwheat leaf genome DNA.
The technical scheme is as follows: the invention provides a method for rapidly extracting buckwheat leaf genome DNA, which comprises the following steps:
(1) putting buckwheat leaves and glass beads into a centrifugal tube, and putting the centrifugal tube into a foam box;
(2) pouring liquid nitrogen into the foam box, covering all centrifuge tubes, sealing the foam box and then shaking to enable internal blades to be powdered;
(3) adding an extracting solution into a centrifugal tube, completely mixing the extracting solution and the leaf powder, carrying out warm bath, adding phenol chloroform isoamylol with the same volume, uniformly mixing, and centrifuging, wherein the extracting solution comprises the following components: ethylenediaminetetraacetic acid (EDTA), Sodium Dodecyl Sulfate (SDS), sodium chloride (NaCl), and Tris (hydroxymethyl) aminomethane (Tris);
(4) centrifuging, collecting supernatant, adding equal volume of precooled isopropanol, mixing, and cooling;
(5) centrifuging the mixed solution, and removing the supernatant;
(6) adding ethanol for cleaning, centrifuging, and removing supernatant;
(7) add ddH to centrifuge tube2Dissolving the buckwheat in the solution O to obtain the buckwheat genome DNA.
Further, in the step (1), the extracting solution is preheated.
Further, the foam box is sealed in the step (2) and then turned upside down and shaken from side to side.
Further, in the step (2), when the height of the centrifugal tube in the foam box reaches half of the depth of the foam box, liquid nitrogen is poured in.
Further, the ethanol concentration in the step (6) is 70%.
Has the advantages that: the method has the advantages of short required time and simple extraction steps, can extract a large amount of buckwheat leaf genome DNA within about 30 minutes to 1 hour, and shortens the time by over 50 percent compared with the traditional CTAB method. The method has the advantages of low price of used equipment and reagents, no need of a special sample grinder, 50% cost saving compared with the traditional CTAB method reagent, suitability for extracting high-flux genome DNA, and suitability for high-flux screening of current molecular marker assisted breeding.
Drawings
FIG. 1 is a diagram showing the extraction results of genomic DNA from buckwheat leaves, in which ddH is present2O as blank control.
Detailed Description
The extraction method of this example is as follows:
the method comprises the following steps of (1) taking buckwheat young leaves with 3-4 leaf ages for extracting genome DNA, or selecting mature leaves for extracting the genome DNA, wherein the method for quickly extracting the buckwheat leaf DNA comprises the following steps:
1. filling about 300mg into a 2ml centrifuge tube, putting 0.5g of small glass beads with the diameter of 0.5mm, and then putting the centrifuge tube into a foam box with the length, width and height of 20 cm; hundreds of centrifugal tubes containing the leaves can be simultaneously placed for batch extraction of buckwheat leaf genome DNA;
2. liquid nitrogen was poured into the foam box, covering all the tubes. Then, after the foam box is sealed, manually shaking the foam box for 5-10 times to enable the internal blades to be powdered;
3. adding 500 μ l of the extract into a 2.0ml centrifuge tube containing the powdered extract (the extract can be preheated in an oven at 65 ℃), putting the centrifuge tube on a 96-hole centrifuge tube frame, turning upside down and shaking left and right to completely mix the extract and the leaf powder, and then putting the centrifuge tube in an oven at 65 ℃ for warm bath for 10-15 min, and shaking and mixing the mixture for several times; then adding phenol chloroform isoamyl alcohol with the same volume, reversing the upside down, forcibly mixing uniformly, and then centrifuging at 12000r/min for 10min (Eppendorf high-speed refrigerated centrifuge);
wherein the formula of the extracting solution is as follows: 9.3g/L of Ethylene Diamine Tetraacetic Acid (EDTA), 5g/L of Sodium Dodecyl Sulfate (SDS), 14.5g/L of sodium chloride (NaCl) and 24.2g/L of Tris (hydroxymethyl) aminomethane (Tris), and the pH is adjusted to 7.5.
Phenol chloroform isoamyl alcohol ratio: 25: 24: 1 (volume ratio)
4. Centrifuging, putting 500-600 mu l of supernatant into a new 1.5ml centrifuge tube, adding equal volume of pre-cooled isopropanol, turning upside down, mixing uniformly, and placing in a refrigerator at-20 ℃ for cold bath for 5 min;
5. centrifuging the mixed solution at 12000r/min for 10min, and removing the supernatant;
6. adding 70% ethanol, cleaning, centrifuging at 5000r/min for 2min, slowly discarding supernatant, and blow drying the centrifuge tube in a fume hood (about 3-5 min);
7. add 50. mu.l ddH to each centrifuge tube2Dissolving the buckwheat in the solution O to obtain the buckwheat genome DNA.
8. The dissolved DNA was electrophoretically detected in a 1% agarose gel, as shown in the figure.
Lanes 1-4 in the electropherogram are markers (DL5000, with band sizes from top to bottom of 5000bp, 3000bp, 2000b, respectivelyp, 1500bp, 1000bp, 750bp, 250bp, 100bp), blank control ddH2The extraction results of O and 2 buckwheat leaf samples show that clear genome DNA bands are obtained from the 2 buckwheat leaf samples, the sizes of the genome DNA bands are complete, degradation is not basically generated, and the expected effect is achieved.
Claims (5)
1. A method for rapidly extracting buckwheat leaf genome DNA is characterized in that: the method comprises the following steps:
(1) putting buckwheat leaves and glass beads into a centrifugal tube, and putting the centrifugal tube into a foam box;
(2) pouring liquid nitrogen into the foam box, covering all centrifuge tubes, sealing the foam box and then shaking to enable internal blades to be powdered;
(3) adding an extracting solution into a centrifugal tube, completely mixing the extracting solution and the leaf powder, carrying out warm bath, adding phenol chloroform isoamylol with the same volume, uniformly mixing, and centrifuging, wherein the extracting solution comprises the following components: ethylenediaminetetraacetic acid (EDTA), Sodium Dodecyl Sulfate (SDS), sodium chloride (NaCl), and Tris (hydroxymethyl) aminomethane (Tris);
(4) centrifuging, collecting supernatant, adding equal volume of precooled isopropanol, mixing, and cooling;
(5) centrifuging the mixed solution, and removing the supernatant;
(6) adding ethanol for cleaning, centrifuging, and removing supernatant;
(7) add ddH to centrifuge tube2Dissolving the buckwheat in the solution O to obtain the buckwheat genome DNA.
2. The method for rapidly extracting genomic DNA of buckwheat leaves according to claim 1, wherein: in the step (1), the extracting solution is preheated.
3. The method for rapidly extracting genomic DNA of buckwheat leaves according to claim 1, wherein: and (3) after the foam box is sealed in the step (2), performing up-down inversion and left-right shaking.
4. The method for rapidly extracting genomic DNA of buckwheat leaves according to claim 1, wherein: and (3) pouring liquid nitrogen when the height of the centrifugal tube in the foam box reaches half of the depth of the foam box in the step (2).
5. The method for rapidly extracting genomic DNA of buckwheat leaves according to claim 1, wherein: the ethanol concentration in the step (6) is 70%.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010830007.1A CN111996187A (en) | 2020-08-17 | 2020-08-17 | Method for rapidly extracting buckwheat leaf genome DNA |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202010830007.1A CN111996187A (en) | 2020-08-17 | 2020-08-17 | Method for rapidly extracting buckwheat leaf genome DNA |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101586102A (en) * | 2009-07-10 | 2009-11-25 | 山东省花生研究所 | Genomic DNA extraction method of peanut leaf blades |
| CN104561284A (en) * | 2014-12-26 | 2015-04-29 | 中国农业科学院棉花研究所 | Molecular identification method for zero-type fruit branch genes of cotton |
| CN104694530A (en) * | 2015-03-10 | 2015-06-10 | 西北农林科技大学 | Extraction method of wheat genome DNA |
| CN105039310A (en) * | 2015-07-28 | 2015-11-11 | 福建师范大学 | Kit for extracting agrobacterium tumefaciens plasmid DNA |
| CN108676862A (en) * | 2018-06-01 | 2018-10-19 | 九江学院 | The identification method of Chinese medicine peach kernel based on fragment length polymorphism PCR |
-
2020
- 2020-08-17 CN CN202010830007.1A patent/CN111996187A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101586102A (en) * | 2009-07-10 | 2009-11-25 | 山东省花生研究所 | Genomic DNA extraction method of peanut leaf blades |
| CN104561284A (en) * | 2014-12-26 | 2015-04-29 | 中国农业科学院棉花研究所 | Molecular identification method for zero-type fruit branch genes of cotton |
| CN104694530A (en) * | 2015-03-10 | 2015-06-10 | 西北农林科技大学 | Extraction method of wheat genome DNA |
| CN105039310A (en) * | 2015-07-28 | 2015-11-11 | 福建师范大学 | Kit for extracting agrobacterium tumefaciens plasmid DNA |
| CN108676862A (en) * | 2018-06-01 | 2018-10-19 | 九江学院 | The identification method of Chinese medicine peach kernel based on fragment length polymorphism PCR |
Non-Patent Citations (3)
| Title |
|---|
| ANASTASIS CHRISTOU等: "Establishment of a rapid, inexpensive protocol for extraction of high quality RNA from small amounts of strawberry plant tissues and other recalcitrant fruit crops", GENE, vol. 537, no. 1, pages 169 - 173, XP028819843, DOI: 10.1016/j.gene.2013.11.066 * |
| 余立辉等: "银杏DNA提取及RAPD分析", 生物技术通报, no. 02, pages 81 - 84 * |
| 郑小伟等主编: "新世纪全国高等中医药院校创新教材 实验中医学", vol. 1, 中国矿业大学出版社, pages: 227 * |
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Application publication date: 20201127 |