CN109439652A - A kind of extracting method of the plant genome DNA rich in polysaccharide - Google Patents
A kind of extracting method of the plant genome DNA rich in polysaccharide Download PDFInfo
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- CN109439652A CN109439652A CN201811393338.2A CN201811393338A CN109439652A CN 109439652 A CN109439652 A CN 109439652A CN 201811393338 A CN201811393338 A CN 201811393338A CN 109439652 A CN109439652 A CN 109439652A
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Abstract
The invention discloses a kind of extracting methods of plant genome DNA rich in polysaccharide, include the following steps: using liquid nitrogen grinding；The CTAB-free buffer of pre-cooling is added, mixing fullys shake in mercaptoethanol；It places on ice, controls temperature at 4 DEG C, centrifugation discards supernatant；It is cleaned once with PBS again according to viscosity, SDS solution is added, mercaptoethanol mixes；Then during which water-bath is mixed by inversion；After cooling, isometric phenol: chloroform is added, mixing of turning upside down；It is centrifuged n, takes supernatant, RNase A is added, mixing of turning upside down is placed at room temperature for；Isopropanol is added, is mixed by inversion, is placed at room temperature for or low temperature is placed, supernatant is abandoned in centrifugation, is cleaned using 75% ethyl alcohol, and the aqua sterilisa dissolution of appropriate volume is added.Method of the invention has better extraction effect for the plant gene rich in polysaccharide, all extracts than conventional method for genomic integrity and subsequent purity more preferable.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to a kind of extraction side of the plant genome DNA rich in polysaccharide
Method.
Background technique
In recent years, since the development of Protocols in Molecular Biology and sequencing technologies is researcher microorganisms genome
Sequence, synthesis mechanism of secondary metabolite etc. provide good basis, and the acquisition of DNA of plants is then the base of these work
Plinth.
Polysaccharide largely exists in plant cell as the structure composition substance and nutritional reservoir substance of plant cell.Polysaccharide
It is the main matter for interfering DNA of plants to extract.The many physicochemical properties and DNA of polysaccharide are much like, therefore are difficult to separate them.
The colloid substance that the combination of polysaccharide and DNA is formed not only influence the operation of experiment and also by cause the activated centre of enzyme change or
Must group change and inhibit the activity of many enzymes, seriously affect the downstream work of molecular biology research.
With the high speed development of sequencing technologies, the SMRT technology of Pacific Bioscience causes that scientific research personnel's is extensive
Concern.The sequencing is also based on the principle being sequenced in synthesis, this technology has used Zero-Mode Wave guide
(ZMW) (zero level waveguide).The process of sequencing: be fluorescently labeled the nucleotide of phosphoric acid group on polymerase active site with template
Chain combination (dye marker that every kind of deoxynucleotide is not had to color), is inspired fluorescence, after fluorescent pulse, is marked
The phosphoric acid group of note is cut and is discharged, and polymerase is transferred to next position, and next deoxynucleotide is connected on site
Start to discharge fluorescent pulse, carries out next circulation.The technology is especially high to the quality of DNA and purity requirement, as DNA degrades
Then cause data volume few, the quality of data is poor, and splicing effect is undesirable；DNA purity is not high, there is the pollution meeting of the substances such as sugar or salt
It causes polymerase activity to reduce even to inactivate, causes the waste of experimental cost.
The previous genome for polysaccharide plant extracts, and can only be extracted using kit, but kit is expensive, passes through
Column also have it is different degrees of interrupt, cause DNA imperfect.This experimental method is simply easily operated, and time-consuming short, cost is relatively low,
Band is complete, the impurity pollution such as Polysaccharide removing that can be clean, suitable for a variety of extractions rich in polysaccharide Plant Genome.
Summary of the invention
In order to overcome drawbacks described above of the existing technology, it is high that the purpose of the present invention is to provide a kind of DNA DNA purities,
The extracting method of the good plant genome DNA rich in polysaccharide of integrality.
In order to realize the purpose of foregoing invention, used technical solution is:
A kind of extracting method of the plant genome DNA rich in polysaccharide, includes the following steps:
30-300S is ground under the range of 55-65Hz using liquid nitrogen；
CTAB-free buffer, the 20ul mercaptoethanol of 1ml pre-cooling is added, mixing fullys shake；
2-3min on ice is placed, temperature is controlled at 4 DEG C, with the speed of 2000-5000rpm, is centrifuged 5-20 minutes, discards
Supernatant；
Cleaning Polysaccharide removing class impurity is carried out using PBS solution；Used PBS solution is 1XPBS solution: i.e. 10XPBS
Solution (Ambion, 500ml/10X PBS buffer PH 7.4, article No.: AM9624) dilutes 10 times with sterile water.
SDS solution 500-1000ul, the 1%-4% mercaptoethanol of 65 DEG C of preheatings is accessed, is mixed；
It is then placed in 20-60min in 65 DEG C of water-baths, is during which at least mixed by inversion 3 times；
After cooling, isometric phenol: chloroform is added, 30-40 mixing of turning upside down；
With the speed of 12000rpm, it is centrifuged 5-15min, takes supernatant, 1-5ul RNase A, mixing of turning upside down, room is added
Temperature places 5-30min；
The isopropanol of 0.6-1 times of volume is added, is mixed by inversion, is placed at room temperature for 10-20min or low temperature placement is small not less than one
When, 10000-12000rpm is centrifuged 10-30 minutes, abandons supernatant；
It is cleaned once using 75% ethyl alcohol, outwells ethyl alcohol, clean to ethyl alcohol volatilization, the sterilizing that appropriate volume is added is water-soluble
Solution.
In a preferred embodiment of the invention, the CTAB-free buffer of the pre-cooling, by densimeter, including such as
Lower component:
0.2M Tris-Hcl；
0.05M EDTA；
0.25M Nacl；
4%PVP；
2% mercaptoethanol is pre-chilled stand-by after configuration is good, in the range of being placed on 4 DEG C.
In a preferred embodiment of the invention, the SDS solution, by densimeter, including following component:
100mM Tris-Hcl (pH=8.0)；
25mM EDTA (pH=8.0)；
500mM Nacl；
1-2%SDS.
In a preferred embodiment of the invention, the temperature that the low temperature is placed is not less than -80 DEG C.
The beneficial effects of the present invention are:
Method of the invention has better extraction effect for the plant gene rich in polysaccharide, in genomic integrity and
It is all extracted than conventional CTAB method or QIAgen DNA extraction kit for subsequent separating degree more preferable.
Detailed description of the invention:
Fig. 1 is the electrophoresis result figure extracted using this experimental method；
Fig. 2 is the electrophoresis result figure extracted using routine CTAB method；
Fig. 3 is the electrophoresis result figure extracted using QIAgen DNA extraction kit.
Specific embodiment
The present invention is further illustrated by the following examples, but these embodiments must not be used to explain to the present invention
Limitation.
On after jasmine petal, robur blade, crape myrtle blade, Chinese catalpa blade, lilac sample are handled in accordance with the following steps
Machine:
1. CTAB-free buffer, the 20ul mercaptoethanol of 1ml pre-cooling is added, mixing fullys shake；
2. placing 2-3min on ice, 4 DEG C, 4000rpm, it is centrifuged 10min, discards supernatant；
3. 1ml PBS is added, mix, 4 DEG C, 4000rpm, is centrifuged 10min, discards supernatant；
4. cleaning the removal be once subject to and reached to polysaccharide impurity with PBS again according to viscosity selection；
5. SDS solution 700ul, the 10ul mercaptoethanol of 65 DEG C of preheatings is added, mix；
6. being put into 25min in 65 DEG C of water-baths, during which at least it is mixed by inversion 3 times；
7. 700ul phenol: chloroform after cooling, is added, 30-40 mixing of turning upside down；
8.12000rpm is centrifuged 5min, takes supernatant (about 700ul), and 2ul RNase A, mixing of turning upside down, room temperature is added
Place 5min；
9. 700ul isopropanol is added, it is mixed by inversion, is placed at room temperature for 10min, 12000rpm, is centrifuged 10min；
10. the cleaning of 75% ethyl alcohol of 1ml is added, discard ethyl alcohol, it is to be evaporated it is clean after, it is heavy that the dissolution of 50ul sterile water is added
It forms sediment；
Wherein, the CTAB-free buffer of pre-cooling are as follows:
0.2M Tris-Hcl；
0.05M EDTA；
0.25M Nacl；
4%PVP；
2% mercaptoethanol (used time single tube adds) is pre-chilled stand-by after configuration is good, in the range of being placed on 4 DEG C.
SDS solution are as follows:
100mM Tris-Hcl (pH=8.0)；
25mM EDTA (pH=8.0)；
500mM Nacl；
1-2%SDS.
Final experimental result is shown in Fig. 1, and in addition Fig. 2 is the electrophoresis result figure using the extraction of routine CTAB method；Fig. 3 is to use
The electrophoresis result figure that QIAgen DNA extraction kit is extracted
Fig. 2 is from electrophoretogram, and impurity is more in electrophoresis hole, and the degradation of DNA traction is serious.
Fig. 3, although impurity is less in used column extraction electrophoresis hole, DNA can be interrupted by crossing column, there is slight traction degradation.
Claims (4)
1. a kind of extracting method of the plant genome DNA rich in polysaccharide, which comprises the steps of:
30-300S is ground under the range of 55-65Hz using liquid nitrogen；
CTAB-free buffer, the 20ul mercaptoethanol of 1ml pre-cooling is added, mixing fullys shake；
2-3min on ice is placed, temperature is controlled at 4 DEG C, with the speed of 2000-5000rpm, is centrifuged 5-20 minutes, discards supernatant；
Cleaning Polysaccharide removing class impurity is carried out using PBS solution；
SDS solution 500-1000ul, the 1%-4% mercaptoethanol of 65 DEG C of preheatings is accessed, is mixed；
It is then placed in 20-60min in 65 DEG C of water-baths, is during which at least mixed by inversion 3 times；
After cooling, isometric phenol: chloroform is added, 30-40 mixing of turning upside down；
With the speed of 12000rpm, it is centrifuged 5-15min, takes supernatant, 1-5ul RNase A, mixing of turning upside down is added, room temperature is put
Set 5-30min；
The isopropanol of 0.6-1 times of volume is added, is mixed by inversion, is placed at room temperature for 10-20min or low temperature was placed not less than one hour,
10000-12000rpm is centrifuged 10-30 minutes, abandons supernatant,；
It is cleaned once using 75% ethyl alcohol, outwells ethyl alcohol, it is clean to ethyl alcohol volatilization, the aqua sterilisa dissolution of appropriate volume is added.
2. a kind of extracting method of the plant genome DNA rich in polysaccharide as described in claim 1, which is characterized in that institute
The CTAB-free buffer for stating pre-cooling, by densimeter, including following component:
0.2M Tris-Hcl；
0.05M EDTA；
0.25M Nacl；
4%PVP；
2% mercaptoethanol is pre-chilled stand-by after configuration is good, in the range of being placed on 4 DEG C.
3. a kind of extracting method of the plant genome DNA rich in polysaccharide as described in claim 1, which is characterized in that institute
SDS solution is stated, by densimeter, including following component:
100mM Tris-Hcl, pH=8.0；
25mM EDTA, pH=8.0；
500mM Nacl；
1-2%SDS.
4. a kind of extracting method of the plant genome DNA rich in polysaccharide as described in claim 1, which is characterized in that institute
The temperature of low temperature placement is stated not less than -80 DEG C.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110804612A (en) * | 2019-12-20 | 2020-02-18 | 福建省农业科学院亚热带农业研究所（福建省农业科学院蔗麻研究中心） | Method for extracting trace and high-quality dendrobium officinale genome DNA |
| CN113736774A (en) * | 2021-07-30 | 2021-12-03 | 贵州光正制药有限责任公司 | Method for extracting DNA from fruit upper leaves |
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| US20070015165A1 (en) * | 2005-07-13 | 2007-01-18 | Sigma-Aldrich Co. | Method for the isolation of RNA from biological sources |
| CN103756994A (en) * | 2013-03-21 | 2014-04-30 | 四川农业大学 | DNA extraction method for polysaccharide-rich plant dried leaves |
| CN104357439A (en) * | 2014-11-27 | 2015-02-18 | 广东省农业科学院作物研究所 | Method for extracting RNA (ribonucleic acid) from plant material containing rich polysaccharides and polyphenols |
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2018
- 2018-11-21 CN CN201811393338.2A patent/CN109439652A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070015165A1 (en) * | 2005-07-13 | 2007-01-18 | Sigma-Aldrich Co. | Method for the isolation of RNA from biological sources |
| CN103756994A (en) * | 2013-03-21 | 2014-04-30 | 四川农业大学 | DNA extraction method for polysaccharide-rich plant dried leaves |
| CN104357439A (en) * | 2014-11-27 | 2015-02-18 | 广东省农业科学院作物研究所 | Method for extracting RNA (ribonucleic acid) from plant material containing rich polysaccharides and polyphenols |
Non-Patent Citations (3)
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| 张子雄等: "柱花草花粉不育和可育分离群体基因组DNA分离方法优化", 《分子植物育种》 * |
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| 陈宗游等: "短序十大功劳基因组总DNA提取方法研究", 《基因组学与应用生物学》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110804612A (en) * | 2019-12-20 | 2020-02-18 | 福建省农业科学院亚热带农业研究所（福建省农业科学院蔗麻研究中心） | Method for extracting trace and high-quality dendrobium officinale genome DNA |
| CN113736774A (en) * | 2021-07-30 | 2021-12-03 | 贵州光正制药有限责任公司 | Method for extracting DNA from fruit upper leaves |
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Application publication date: 20190308 |