CN105695446B - A method of extracting soya seeds complete genome DNA - Google Patents
A method of extracting soya seeds complete genome DNA Download PDFInfo
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- CN105695446B CN105695446B CN201610099160.5A CN201610099160A CN105695446B CN 105695446 B CN105695446 B CN 105695446B CN 201610099160 A CN201610099160 A CN 201610099160A CN 105695446 B CN105695446 B CN 105695446B
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Abstract
The invention discloses a kind of methods for extracting soya seeds complete genome DNA, the key step of this method are as follows: nucleus extraction buffer is added into the soybean sample that liquid nitrogen is fully ground, by Pintsch process, phenol/chloroform and chloroform/isoamyl alcohol extraction, protease digestion, RNA enzyme processing, isopropanol precipitating and ethanol washing, the soybean complete genome DNA of rapidly extracting to higher concentration and purity.The more traditional DNA extraction method of the present invention (CTAB method) used time greatly shortens, and DNA mass also improves；Compared with DNA extraction kit method, DNA concentration is doubled, and expense but substantially reduces, and the used time is suitable.The present invention is suitable for being stored at room temperature and the extraction of fresh soya seeds high quality complete genome DNA.
Description
Technical field
The invention belongs to molecular biology fields, and in particular to a method of extract soya seeds complete genome DNA.
Background technique
With the fast development of molecular biology, a large amount of fundamental biological knowledge research is carried out both at home and abroad and molecule is auxiliary
Help breeding, extracting genome DNA as in molecule experiments the most basis and important technological means is applied in each research field.
Many researchs need many experiments material, and DNA extracts heavy workload, and purity requirement is high, and the extraction quality and efficiency of DNA will
It will have a direct impact on subsequent experimental result.
Soybean (scientific name: Glycine max (L.) Merr) is generally called soya bean.Belong to Dicotyledoneae, beans mesh, papilionaceous flower
Section, Glycine annual herb plant.Nutritive value is most abundant, the protein containing high-quality about 40%.Fat content is 15%
To 20%, it is rich in unsaturated fatty acid.In addition, also containing the minerals such as vitamin A, B, D, E and calcium, phosphorus, iron.Just because of big
Albumen and fatty equal size are abundant in beans, the difficulty extracted compared with other staple crops (such as rice, wheat, corn etc.) DNA compared with
It is high.Time-consuming for the extraction such as traditional CTAB method, SDS method or Low pH extraction with high salts method genomic DNA, and extracts the full base of soya seeds
Impurity when because of group DNA is relatively more；And the soya seeds complete genome DNA concentration that kit method extracts is not also high, Wu Faman
Sufficient genomics research.Therefore it is badly in need of a kind of easy, efficiently, economic method for extracting soya seeds complete genome DNA.
Summary of the invention
The purpose of the present invention is to provide a kind of methods for extracting soya seeds complete genome DNA.
The purpose of the present invention can be achieved through the following technical solutions:
A method of extracting soya seeds complete genome DNA, comprising the following steps:
(1) soya seeds liquid feeding nitrogen is ground into fine powder；
(2) 90~95 DEG C of water after addition cell extraction buffer and 0.2mol/L NaOH are mixed into the fine powder after grinding
8-10min is bathed, is then centrifuged for staying supernatant；
(3) it is added the Proteinase K and 10mg/ml RNAase of 20mg/mL into step (2) obtained supernatant, 56 DEG C
Water-bath 10min is then centrifuged for staying supernatant；
(4) mixed liquor with supernatant isometric phenol and chloroform, institute are added into step (3) obtained supernatant
Stating phenol and chloroform volume ratio in mixed liquor is 1:1, mixes well and takes supernatant after being centrifuged；
(5) mixed liquor with supernatant isometric chloroform and isoamyl alcohol is added into step (4) obtained supernatant,
Chloroform and isoamyl alcohol volume ratio are 24:1 in the mixed liquor, mix well and take supernatant after being centrifuged；
(6) isopropanol of 4/5 volume of supernatant is added into step (5) obtained supernatant, is precipitated in -20 DEG C
Supernatant, the DNA precipitated are abandoned in centrifugation afterwards；
(7) 75% (V/V) ethanol washing is added into step (6) obtained DNA, centrifugation is abandoned supernatant, precipitated
DNA；
(8) dehydrated alcohol washing is added into the obtained precipitating DNA of step (7), supernatant, remaining ethyl alcohol are abandoned in centrifugation
Volatilization is clean, and the dissolution of TE solution is added, saves backup at -20~-70 DEG C.
The cell extraction buffer ingredient are as follows: 0.01mol/L Tris, 1mmol/L EDTA, 0.1mol/LNaCl,
PH is 8.0.
The TE solution composition are as follows: 10mmol/L Tris-HCl, 1mmol/L EDTA, pH 8.0.
The amount ratio of fine powder and cell extraction buffer and 0.2mol/L NaOH in step (2) after grinding are as follows: every 50mg
880 μ L cell extraction buffers, 120 μ L 0.2mol/L NaOH are added in fine powder after grinding.
The additive amount of Proteinase K and RNAase in step (3) are as follows: 10 μ L Proteinase K (20mg/ are added in 2.0mL centrifuge tube
) and 5 μ L RNAase (10mg/mL) mL.
The condition of centrifugation described in step (2) are as follows: 12 000r/min are centrifuged 5~10min.
The condition of centrifugation described in step (3)~(8) are as follows: 12 000r/min are centrifuged 10~15min.
The time of precipitating described in step (6) is 10~15min.
The present invention provides a kind of quick, economic method for extracting soya seeds complete genome DNA, is related to molecular biology neck
Plant process for extracting complete genome DNA in domain.This method has main steps that: being added into the soybean sample that liquid nitrogen is fully ground thin
Karyon Extraction buffer, by Pintsch process, protease digestion, RNA enzyme is handled, phenol/chloroform and chloroform/isoamyl alcohol extraction,
The soybean complete genome DNA of isopropanol precipitating and ethanol washing, rapidly extracting to higher concentration and purity.The present invention is more traditional
DNA extraction method (CTAB method) used time greatly shorten, DNA mass also improves；Compared with DNA extraction kit method,
DNA concentration doubles, and expense but substantially reduces, and the used time is suitable.The present invention is suitable for being stored at room temperature and fresh Soybean Species
The extraction of sub- high quality complete genome DNA.
Most preferably detailed technology scheme of the invention are as follows:
(1) soya seeds are weighed, is placed in mortar and liquid nitrogen grinding is added into fine powder；
(2) soybean fine powder after grinding is transferred in 2.0mL centrifuge tube, every pipe 50mg, it is slow adds 880 μ L cell extractions
Fliud flushing, 120 μ L 0.2mol/L NaOH, 95 DEG C of water-baths 8min, 12 000r/min are centrifuged 5min after mixing, and Aspirate supernatant adds
Enter in new 2.0mL centrifuge tube；Cell extraction buffer ingredient are as follows: 1mL 1mol/L Tris, 0.2mL 0.5mol/L EDTA,
0.5844g NaCl is settled to 100mL, pH 8.0；
(3) it is added 10 μ L Proteinase Ks (20mg/mL) and 56 DEG C of 5 μ l RNAase (10mg/mL) water-bath 10min, 12
000r/min is centrifuged 10min, and Aspirate supernatant is added in new 2.0mL centrifuge tube；
(4) isometric phenol/chloroform (1:1) is added, mixes well, 12 000r/min are centrifuged 10min, draw supernatant
Liquid is added in new 2.0mL centrifuge tube；
(5) the isometric chloroform/isoamyl alcohol (24:1) of addition, mixes well, 12 000r/min centrifugation 10min, in absorption
Clear liquid is added in new 2.0mL centrifuge tube；
(6) isopropanol of 4/5 volume is added in supernatant, -20 DEG C of precipitatings 10min, 12 000r/min are centrifuged 10min, abandon
Supernatant, the DNA precipitated；
(7) 1mL 75% ethanol washing DNA, 12 000r/min is added and is centrifuged 2min, abandons supernatant, is precipitated
DNA；
(8) washing of 500 μ L dehydrated alcohols is added, abandons supernatant, remaining ethyl alcohol volatilization is clean；
50 μ L TE dissolution is added, is saved backup at -20~-70 DEG C；TE solution composition are as follows: 10 mmol/L Tris-
HCl, 1mmol/L EDTA, pH 8.0.
The DNA solution for taking 2 μ l above-mentioned steps to prepare is detected through 1% agarose gel electrophoresis, and in nucleic acid-protein detector
Upper detection A260nm/A280nmAnd DNA concentration.
Beneficial effects of the present invention:
The time can be shortened to 1.5h by extracting soya seeds complete genome DNA using this experimental method, test agents useful for same
It is less, relatively universal, and experiment flow is fairly simple.
Detailed description of the invention
Fig. 1 is the gel electrophoresis of soybean complete genome DNA.
In figure: 1 representative extracts fresh soyabean seed with kit, and 2 representative kits extract the Soybean Species being stored at room temperature
Son, 3 represent with present invention extraction fresh soyabean seed, the soya seeds that 4 representatives are stored at room temperature with present invention extraction, and M is
DNAMarker。
Specific embodiment
Technical solution of the present invention is further illustrated in conjunction with the drawings and specific embodiments
Embodiment 1
(1) weigh respectively 100mg be stored at room temperature with fresh soya seeds, be respectively placed in mortar and liquid nitrogen be added, sufficiently
It is ground into fine powder；
(2) soybean fine powder after grinding is transferred in 2.0mL centrifuge tube, every pipe 50mg, it is slow adds 880 μ L cell extractions
Fliud flushing (1mL 1mol/L Tris, 0.2mL 0.5mol/L EDTA, 0.5844g NaCl are settled to 100mL), 120 μ L
0.2mol/L NaOH；95 DEG C of water-baths 8min, 12 000r/min are centrifuged 5min after mixing, and careful Aspirate supernatant is added new
In 2.0ml centrifuge tube；
(3) it is added 10 μ L Proteinase Ks (20mg/mL) and 56 DEG C of 5 μ l RNAase (10mg/mL) water-bath 10min, 12
000r/min is centrifuged 10min, and careful Aspirate supernatant is added in new 2.0mL centrifuge tube；
(4) isometric phenol/chloroform (1:1) is added, mixes well, 12 000r/min are centrifuged 10min, careful to draw
Supernatant is added in new 2.0mL centrifuge tube；
(5) isometric chloroform/isoamyl alcohol (24:1) is added, mixes well, 12 000r/min are centrifuged 10min, careful to inhale
Supernatant is taken to be added in new 2.0mL centrifuge tube；
(6) isopropanol of 4/5 volume is added into supernatant, mixes well, -20 DEG C of precipitatings 10min, 12 000r/min
It is centrifuged 10min, abandons supernatant, the DNA precipitated；
(7) 1mL 75% ethanol washing DNA, 12 000r/min is added and is centrifuged 2min, abandons supernatant, is precipitated
DNA；
(8) washing of 500 μ L dehydrated alcohols is added, abandons supernatant, superclean bench drying；50 μ L TE (10mmol/L are added
Tris-HCl, 1mmol/L EDTA, pH 8.0) dissolution, it is saved backup at -20 DEG C.
Comparative example
It is pressed using the plant genome DNA extracts kit (DP305) that TIANGEN Biotech (Beijing) Co., Ltd. produces
Extract the DNA being stored at room temperature with fresh soya seeds respectively as directed.
The DNA solution for taking 2 μ L above-described embodiments and comparative example to prepare, is detected through 1% agarose gel electrophoresis, in Fig. 1
Tetra- band of 1-4 is respectively from left to right: the fresh soyabean seed extracted with kit, big with being stored at room temperature for kit extraction
Beans seed, the fresh soyabean seed extracted with the present invention, the soya seeds being stored at room temperature extracted with the present invention；M is
DNAMarker.OD is detected on nucleic acid-protein detector260nm/OD280nmAnd DNA concentration result such as table 1.
Table 1 extracts gained soybean complete genome DNA concentration and purity
Note: 1 representative extracts fresh soyabean seed with kit in table, and 2 representative kits extract the soybean being stored at room temperature
Seed, 3 represent with present invention extraction fresh soyabean seed, the soya seeds that 4 representatives are stored at room temperature with present invention extraction.
In addition, embodiment 1, which extracts soya seeds DNA, only needs 1.5h, Soybean Species are extracted using CTAB method in the prior art
Sub- DNA generally requires 3 hours, and extraction time shortens half compared with existing CTAB method, and DNA mass also improves.
The time can be shortened to 1.5h by extracting soya seeds complete genome DNA using this experimental method, test agents useful for same
It is less, relatively universal, and experiment flow is fairly simple, it is easier to it operates, the DNA band extracted compared with comparative example is apparent, pure
Degree and concentration are higher (DNA concentration doubles), and expense but substantially reduces, and the used time is substantially suitable.Kit and new method will be used
The DNA of extraction is all satisfied molecule experiments requirement after PCR is verified, but the band amplified has no significant difference.
Claims (7)
1. a kind of method for extracting soya seeds complete genome DNA, it is characterised in that the following steps are included:
(1) soya seeds liquid feeding nitrogen is ground into fine powder；
(2) 90~95 DEG C of water-bath 8- after addition cell extraction buffer and 0.2mol/L NaOH are mixed into the fine powder after grinding
10min is then centrifuged for staying supernatant；
(3) Proteinase K and 10mg/ml RNAase of 20mg/mL, 56 DEG C of water-baths are added into step (2) obtained supernatant
10min is then centrifuged for staying supernatant；
(4) mixed liquor with supernatant isometric phenol and chloroform is added into step (3) obtained supernatant, it is described mixed
Closing phenol and chloroform volume ratio in liquid is 1:1, mixes well and takes supernatant after being centrifuged；
(5) mixed liquor with supernatant isometric chloroform and isoamyl alcohol is added into step (4) obtained supernatant, it is described
Chloroform and isoamyl alcohol volume ratio are 24:1 in mixed liquor, mix well and take supernatant after being centrifuged；
(6) isopropanol of 4/5 volume of supernatant is added into step (5) obtained supernatant, in -20 DEG C precipitating after from
The heart abandons supernatant, the DNA precipitated；
(7) 75% ethanol washing is added into step (6) obtained DNA, supernatant, the DNA precipitated are abandoned in centrifugation；
(8) dehydrated alcohol washing is added into the obtained precipitating DNA of step (7), supernatant, remaining ethyl alcohol volatilization are abandoned in centrifugation
Completely, the dissolution of TE solution is added, is saved backup at -20~-70 DEG C；
The cell extraction buffer ingredient are as follows: 0.01mol/L Tris, 1mmol/L EDTA, 0.1mol/LNaCl, pH are
8.0。
2. it is according to claim 1 extract soya seeds complete genome DNA method, it is characterised in that the TE solution at
It is divided into: 10mmol/L Tris-HCl, 1mmol/L EDTA, pH 8.0.
3. the method according to claim 1 for extracting soya seeds complete genome DNA, it is characterised in that step is ground in (2)
The amount ratio of fine powder and cell extraction buffer and 0.2mol/L NaOH after mill are as follows: be added in the fine powder after every 50mg grinding
880 μ L cell extraction buffers, 120 μ L 0.2mol/L NaOH.
4. the method according to claim 1 for extracting soya seeds complete genome DNA, it is characterised in that egg in step (3)
The additive amount of white enzyme K and RNAase are as follows: 10 μ L 20mg/mL Proteinase Ks and 5 μ L 10mg/mL are added in 2.0mL centrifuge tube
RNAase。
5. the method according to claim 1 for extracting soya seeds complete genome DNA, it is characterised in that institute in step (2)
State the condition of centrifugation are as follows: 12 000r/min are centrifuged 5~10min.
6. the method according to claim 1 for extracting soya seeds complete genome DNA, it is characterised in that step (3)~(8)
Described in centrifugation condition are as follows: 12 000r/min be centrifuged 10~15min.
7. the method according to claim 1 for extracting soya seeds complete genome DNA, it is characterised in that institute in step (6)
The time for stating precipitating is 10~15min.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102220313A (en) * | 2011-05-31 | 2011-10-19 | 广西作物遗传改良生物技术重点开放实验室 | Gene group DNA extraction method suitable for crop SSR (Single Sequence Repeats) molecular marker analysis |
| CN104263719A (en) * | 2014-01-23 | 2015-01-07 | 深圳市圣西马生物技术有限公司 | Oat seed DNA large-scale extraction and purification method suitable for PCR amplification |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102220313A (en) * | 2011-05-31 | 2011-10-19 | 广西作物遗传改良生物技术重点开放实验室 | Gene group DNA extraction method suitable for crop SSR (Single Sequence Repeats) molecular marker analysis |
| CN104263719A (en) * | 2014-01-23 | 2015-01-07 | 深圳市圣西马生物技术有限公司 | Oat seed DNA large-scale extraction and purification method suitable for PCR amplification |
Non-Patent Citations (1)
| Title |
|---|
| 种子DNA提取方法;wchlqld;《https://wenku.baidu.com/view/335ad9b6b8f67clcfbd6b815.html》;20140916;全文 |
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