CN105695446A - Method for extracting soybean seed whole-genome DNA - Google Patents
Method for extracting soybean seed whole-genome DNA Download PDFInfo
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- CN105695446A CN105695446A CN201610099160.5A CN201610099160A CN105695446A CN 105695446 A CN105695446 A CN 105695446A CN 201610099160 A CN201610099160 A CN 201610099160A CN 105695446 A CN105695446 A CN 105695446A
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Abstract
The invention discloses a method for extracting soybean seed whole-genome DNA. The method mainly includes: adding nucleus extraction buffering liquid into a soybean sample fully ground through liquid nitrogen, and quickly extracting the soybean seed whole-genome DNA high in concentration and purity through high-temperature pyrolysis, phenol/chloroform and chloroform/isoamylol extraction, protease digestion, RNA enzyme treatment and isopropanol precipitation and alcohol washing. Compared with conventional DNA extraction methods (CTAB methods), the method has the advantages that time consumption is greatly reduced, and DNA quality is improved to certain extent; compared with a DNA extraction kit method, the method has the advantages that DNA concentration is doubled, cost is lowered greatly, and time consumption is equivalent. The method is suitable for normal-temperature storage and extraction high-quality whole-genome DNA of fresh soybean seeds.
Description
Technical field
The invention belongs to biology field, be specifically related to a kind of method extracting soybean seed complete genome DNA。
Background technology
Along with molecular biological fast development, carried out substantial amounts of fundamental biological knowledge research and marker assisted selection both at home and abroad, extracting genome DNA as in molecule experiments the most basis and important technological means is applied in each research field。A lot of researchs need great many of experiments material, and DNA extraction workload is big, and purity requirement is high, and the extraction quality of DNA and efficiency will directly affect subsequent experimental result。
Semen sojae atricolor (formal name used at school: Glycinemax (L.) Merr) is generally called Semen Glycines。Belong to Dicotyledoneae, bean order, Papilionaceae, Glycine annual herb plant。Nutritive value is the abundantest, the protein containing high-quality about 40%。Fat content is 15% to 20%, rich in unsaturated fatty acid。Additionally, possibly together with mineral such as vitamin A, B, D, E and calcium, phosphorus, ferrum。Enriching just because of albumen in Semen sojae atricolor and fat equal size, relatively the difficulty of other staple crops (such as Oryza sativa L., Semen Tritici aestivi, Semen Maydis etc.) DNA extraction is higher。Traditional CTAB method, SDS method or Low pH extraction with high salts method etc. extract genomic DNA length consuming time, and impurity when extracting soybean seed complete genome DNA is relatively more；And the soybean seed complete genome DNA concentration of kit method extraction is not high yet, it is impossible to meet genomics research。Therefore a kind of simplicity, efficient, the economic method extracting soybean seed complete genome DNA it are badly in need of。
Summary of the invention
It is an object of the invention to provide a kind of method extracting soybean seed complete genome DNA。
The purpose of the present invention can be achieved through the following technical solutions:
A kind of method extracting soybean seed complete genome DNA, comprises the following steps:
(1) soybean seed is added liquid nitrogen and grind to form fine powder；
(2) 90～95 DEG C of water-bath 8-10min after mixing to addition cell extraction buffer in the fine powder after grinding and 0.2mol/LNaOH, are then centrifuged for staying supernatant；
(3) in the obtained supernatant of step (2), add E.C. 3.4.21.64 and the 10mg/mlRNAase of 20mg/mL, 56 DEG C of water-bath 10min, be then centrifuged for staying supernatant；
(4) adding the mixed liquor with the isopyknic phenol of supernatant and chloroform in the obtained supernatant of step (3), in described mixed liquor, phenol and chloroform volume ratio are 1:1, fully mixing also centrifugal after take supernatant；
(5) adding the mixed liquor with the isopyknic chloroform of supernatant and isoamyl alcohol in the obtained supernatant of step (4), in described mixed liquor, chloroform and isoamyl alcohol volume ratio are 24:1, fully mixing also centrifugal after take supernatant；
(6) in the obtained supernatant of step (5), add the isopropanol of described supernatant 4/5 volume, centrifugal after precipitating in-20 DEG C abandon supernatant, the DNA being precipitated；
(7) in the obtained DNA of step (6), add 75% (V/V) washing with alcohol, centrifugal abandon supernatant, the DNA being precipitated；
(8) adding absolute ethanol washing in the obtained precipitation DNA of step (7), centrifugal abandon supernatant, the ethanol volatilization of residual is clean, adds TE solution and dissolves, saves backup at-20～-70 DEG C。
Described cell extraction buffer composition is: 0.01mol/LTris, 1mmol/LEDTA, 0.1mol/LNaCl, pH are 8.0。
Described TE solution composition is: 10mmol/LTris-HCl, 1mmol/LEDTA, pH8.0。
Fine powder after grinding in step (2) with the amount ratio of cell extraction buffer and 0.2mol/LNaOH is: add 880 μ L cell extraction buffer, 120 μ L0.2mol/LNaOH in the fine powder after every 50mg grinding。
In step (3), the addition of E.C. 3.4.21.64 and RNAase is: add 10 μ L E.C. 3.4.21.64 (20mg/mL) and 5 μ LRNAase (10mg/mL) in 2.0mL centrifuge tube。
Condition centrifugal described in step (2) is: 12000r/min is centrifuged 5～10min。
Condition centrifugal described in step (3)～(8) is: 12000r/min is centrifuged 10～15min。
Described in step (6), the time of precipitation is 10～15min。
The present invention provides a kind of method extracting soybean seed complete genome DNA quick, economic, relates to plant process for extracting complete genome DNA in biology field。The method has main steps that: add nucleus extraction buffer in the soybean sample that liquid nitrogen is fully ground, through Pintsch process, protease digestion, RNase processes, phenol/chloroform and chloroform/isoamyl alcohol extraction, isopropanol precipitating and washing with alcohol, rapid extraction is to the Semen sojae atricolor complete genome DNA of higher concentration and purity。DNA extraction method (CTAB method) used time that the present invention is more traditional shortens greatly, and DNA mass also improves；Relatively DNA extraction kit method, DNA concentration doubles, and expense is but substantially reduced, and the used time is suitable。The present invention is applicable to the extraction of room temperature preservation and fresh soybean seed high-quality complete genome DNA。
The most preferably detailed technology scheme of the present invention is:
(1) weigh soybean seed, be placed in mortar and add liquid nitrogen grinding become fine powder；
(2) soybean fine powder after grinding is proceeded in 2.0mL centrifuge tube, often pipe 50mg, adds 880 μ L cell extraction buffer, 120 μ L0.2mol/LNaOH, mixes rear 95 DEG C of water-bath 8min, 12000r/min is centrifuged 5min, and Aspirate supernatant adds in new 2.0mL centrifuge tube；Cell extraction buffer composition is: it is 8.0 that 1mL1mol/LTris, 0.2mL0.5mol/LEDTA, 0.5844gNaCl are settled to 100mL, pH；
(3) adding the centrifugal 10min of 10 μ L E.C. 3.4.21.64 (20mg/mL) and 5 μ lRNAase (10mg/mL) 56 DEG C of water-baths 10min, 12000r/min, Aspirate supernatant adds in new 2.0mL centrifuge tube；
(4) adding isopyknic phenol/chloroform (1:1), fully mix, 12000r/min is centrifuged 10min, and Aspirate supernatant adds in new 2.0mL centrifuge tube；
(5) adding isopyknic chloroform/isoamyl alcohol (24:1), fully mix, 12000r/min is centrifuged 10min, and Aspirate supernatant adds in new 2.0mL centrifuge tube；
(6) supernatant adds the isopropanol of 4/5 volume ,-20 DEG C of centrifugal 10min of precipitation 10min, 12000r/min, abandon supernatant, the DNA being precipitated；
(7) add the centrifugal 2min of 1mL75% washing with alcohol DNA, 12000r/min, abandon supernatant, the DNA being precipitated；
(8) adding 500 μ L absolute ethanol washings, abandon supernatant, the ethanol volatilization of residual is clean；
Add 50 μ LTE to dissolve, save backup at-20～-70 DEG C；TE solution composition is: 10mmol/LTris-HCl, 1mmol/LEDTA, pH8.0。
Take DNA solution prepared by 2 μ l above-mentioned steps, detect through 1% agarose gel electrophoresis, and on nucleic acid-protein detector, detect A260nm/A280nmAnd DNA concentration。
Beneficial effects of the present invention:
Adopting this experimental technique to extract soybean seed complete genome DNA and can shorten the time to 1.5h, experiment agents useful for same is less, relatively general, and experiment flow is fairly simple。
Accompanying drawing explanation
Fig. 1 is Semen sojae atricolor complete genome DNA gel electrophoresis。
In figure: 1 representative test kit extracts fresh soyabean seed, 2 representatives test kit extracts the soybean seed that room temperature preserves, and the 3 representative present invention extract fresh soyabean seed, and the 4 representative present invention extract the soybean seed that room temperature preserves, and M is DNAMarker。
Detailed description of the invention
In conjunction with the drawings and specific embodiments, technical scheme is further illustrated
Embodiment 1
(1) weigh 100mg room temperature respectively to preserve and fresh soybean seed, be respectively placed in mortar and add liquid nitrogen, be fully ground into fine powder；
(2) soybean fine powder after grinding is proceeded in 2.0mL centrifuge tube, often pipe 50mg, add 880 μ L cell extraction buffer (1mL1mol/LTris, 0.2mL0.5mol/LEDTA, 0.5844gNaCl are settled to 100mL), 120 μ L0.2mol/LNaOH；Mixing the centrifugal 5min of rear 95 DEG C of water-baths 8min, 12000r/min, careful Aspirate supernatant adds in new 2.0ml centrifuge tube；
(3) adding the centrifugal 10min of 10 μ L E.C. 3.4.21.64 (20mg/mL) and 5 μ lRNAase (10mg/mL) 56 DEG C of water-baths 10min, 12000r/min, careful Aspirate supernatant adds in new 2.0mL centrifuge tube；
(4) adding isopyknic phenol/chloroform (1:1), fully mix, 12000r/min is centrifuged 10min, and careful Aspirate supernatant adds in new 2.0mL centrifuge tube；
(5) adding isopyknic chloroform/isoamyl alcohol (24:1), fully mix, 12000r/min is centrifuged 10min, and careful Aspirate supernatant adds in new 2.0mL centrifuge tube；
(6) in supernatant, add the isopropanol of 4/5 volume, fully mix ,-20 DEG C of centrifugal 10min of precipitation 10min, 12000r/min, abandon supernatant, the DNA being precipitated；
(7) add the centrifugal 2min of 1mL75% washing with alcohol DNA, 12000r/min, abandon supernatant, the DNA being precipitated；
(8) adding 500 μ L absolute ethanol washings, abandon supernatant, superclean bench dries up；Add 50 μ LTE (10mmol/LTris-HCl, 1mmol/LEDTA, pH8.0) to dissolve, save backup at-20 DEG C。
Comparative example
The plant genome DNA adopting TIANGEN Biotech's production extracts test kit (DP305) according to illustrating that extracting room temperature respectively preserves and the DNA of fresh soybean seed。
Take 2 μ L above-described embodiments and DNA solution prepared by comparative example, detect through 1% agarose gel electrophoresis, 1-4 tetra-band in Fig. 1 is from left to right respectively: the fresh soyabean seed extracted with test kit, the soybean seed that the room temperature extracted with test kit preserves, the fresh soyabean seed extracted by the present invention, the soybean seed that the room temperature extracted by the present invention preserves；M is DNAMarker。Nucleic acid-protein detector detects OD260nm/OD280nmAnd DNA concentration result such as table 1。
Table 1 extracts gained Semen sojae atricolor complete genome DNA concentration and purity
Note: in table, 1 representative test kit extracts fresh soyabean seed, 2 representatives test kit extracts the soybean seed that room temperature preserves, and the 3 representative present invention extract fresh soyabean seed, and the 4 representative present invention extract the soybean seed that room temperature preserves。
It addition, embodiment 1 extracts soybean seed DNA has only to 1.5h, adopting CTAB method to extract soybean seed DNA it is generally required to 3 hours in prior art, extraction time shortens half compared with existing CTAB method, and DNA mass also improves。
Adopt this experimental technique to extract soybean seed complete genome DNA and can shorten the time to 1.5h, experiment agents useful for same is less, more general, and experiment flow is fairly simple, it is easier to operate to, the DNA band of extraction compared with comparative example is apparent, purity and concentration higher (DNA concentration doubles), expense is but substantially reduced, and the used time is substantially suitable。The DNA extracted by test kit and new method is satisfied by molecule experiments requirement after PCR verifies, but the band amplified there is no significant difference。
Claims (8)
1. the method extracting soybean seed complete genome DNA, it is characterised in that comprise the following steps:
(1) soybean seed is added liquid nitrogen and grind to form fine powder；
(2) 90～95 DEG C of water-bath 8-10min after mixing to addition cell extraction buffer in the fine powder after grinding and 0.2mol/LNaOH, are then centrifuged for staying supernatant；
(3) in the obtained supernatant of step (2), add E.C. 3.4.21.64 and the 10mg/mlRNAase of 20mg/mL, 56 DEG C of water-bath 10min, be then centrifuged for staying supernatant；
(4) adding the mixed liquor with the isopyknic phenol of supernatant and chloroform in the obtained supernatant of step (3), in described mixed liquor, phenol and chloroform volume ratio are 1:1, fully mixing also centrifugal after take supernatant；
(5) adding the mixed liquor with the isopyknic chloroform of supernatant and isoamyl alcohol in the obtained supernatant of step (4), in described mixed liquor, chloroform and isoamyl alcohol volume ratio are 24:1, fully mixing also centrifugal after take supernatant；
(6) in the obtained supernatant of step (5), add the isopropanol of described supernatant 4/5 volume, centrifugal after precipitating in-20 DEG C abandon supernatant, the DNA being precipitated；
(7) in the obtained DNA of step (6), add 75% washing with alcohol, centrifugal abandon supernatant, the DNA being precipitated；
(8) adding absolute ethanol washing in the obtained precipitation DNA of step (7), centrifugal abandon supernatant, the ethanol volatilization of residual is clean, adds TE solution and dissolves, saves backup at-20～-70 DEG C。
2. the method for extraction soybean seed complete genome DNA according to claim 1, it is characterised in that described cell extraction buffer composition is: 0.01mol/LTris, 1mmol/LEDTA, 0.1mol/LNaCl, pH are 8.0。
3. the method for extraction soybean seed complete genome DNA according to claim 1, it is characterised in that described TE solution composition is: 10mmol/LTris-HCl, 1mmol/LEDTA, pH8.0。
4. the method for extraction soybean seed complete genome DNA according to claim 1, it is characterised in that the fine powder after grinding in step (2) with the amount ratio of cell extraction buffer and 0.2mol/LNaOH is: add 880 μ L cell extraction buffer, 120 μ L0.2mol/LNaOH in the fine powder after every 50mg grinding。
5. the method for extraction soybean seed complete genome DNA according to claim 1, it is characterised in that in step (3), the addition of E.C. 3.4.21.64 and RNAase is: add 10 μ L20mg/mL E.C. 3.4.21.64s and 5 μ L10mg/mLRNAase in 2.0mL centrifuge tube。
6. the method for extraction soybean seed complete genome DNA according to claim 1, it is characterised in that condition centrifugal described in step (2) is: 12000r/min is centrifuged 5～10min。
7. the method for extraction soybean seed complete genome DNA according to claim 1, it is characterised in that condition centrifugal described in step (3)～(8) is: 12000r/min is centrifuged 10～15min。
8. the method for extraction soybean seed complete genome DNA according to claim 1, it is characterised in that described in step (6), the time of precipitation is 10～15min。
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110734906A (en) * | 2018-07-19 | 2020-01-31 | 浙江大学 | Quick extraction method of nucleic acid of kinds of soybeans and products thereof |
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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| Publication number | Priority date | Publication date | Assignee | Title |
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| 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)
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| WCHLQLD: "种子DNA提取方法", 《HTTPS://WENKU.BAIDU.COM/VIEW/335AD9B6B8F67CLCFBD6B815.HTML》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110734906A (en) * | 2018-07-19 | 2020-01-31 | 浙江大学 | Quick extraction method of nucleic acid of kinds of soybeans and products thereof |
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