CN109628443A - A method of extracting polysaccharide polyphenol plant total serum IgE - Google Patents
A method of extracting polysaccharide polyphenol plant total serum IgE Download PDFInfo
- Publication number
- CN109628443A CN109628443A CN201910142165.5A CN201910142165A CN109628443A CN 109628443 A CN109628443 A CN 109628443A CN 201910142165 A CN201910142165 A CN 201910142165A CN 109628443 A CN109628443 A CN 109628443A
- Authority
- CN
- China
- Prior art keywords
- added
- 5min
- supernatant
- total serum
- serum ige
- 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
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Crystallography & Structural Chemistry (AREA)
- Plant Pathology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a kind of methods for extracting polysaccharide polyphenol plant total serum IgE, Trizol solution is replaced to crack plant tissue with the lysate and beta -mercaptoethanol solution for being directed to polysaccharide polyphenol plant, and water-saturated phenol is added and reduces being mixed into for DNA, while the oxidative damage that beta -mercaptoethanol reduction oxygen generates cell is added.The present invention eliminates preparation and the sterilization process of solution compared with CTAB method, the time is greatly reduced, and can effectively avoid the degradation of carbohydrate and phenols to total serum IgE in plant, while the protein and DNA that can be mixed in fully erased RNA.The extracted total serum IgE of this method can satisfy the research to Micro RNA and its target gene.
Description
Technical field
The present invention relates to field of biology, and in particular to a method of extract polysaccharide polyphenol plant total serum IgE.
Background technique
RNA is very important biomolecule during gene expression, and isolating and purifying to it is the weight for studying gene function
Want one of basis.The RNA of high quality constructs follow-up library, RNA-seq and other molecular biological analysis are extremely heavy
It wants.And for most plants tissue, the RNA for extracting high quality is particularly important, because of most vegetable materials
Rich in polysaccharide, polyphenol, protein and secondary metabolite.Polyphenols is easily oxidized during the grinding process, and energy
Conjugated protein and nucleic acid form macromolecular substances；During the extraction process, polysaccharide and polyphenols may result in RNA drop
Solution interferes subsequent experimental applications, and the method that common Trizol method extracts plant total serum IgE cannot remove the carbohydrate in plant
And phenols.
Summary of the invention
To solve the above problems, the present invention provides a kind of method for extracting polysaccharide polyphenol plant total serum IgE, with for polysaccharide
The lysate of polyphenol plant replaces Trizol solution to crack plant tissue, and water-saturated phenol is added and reduces being mixed into for DNA,
Beta -mercaptoethanol is added simultaneously reduces the oxidative damage that oxygen generates cell.
To achieve the above object, the technical scheme adopted by the invention is as follows:
A method of polysaccharide polyphenol plant total serum IgE is extracted, is included the following steps:
S1, after taking appropriate tissue sample to be fully ground 3~5 times with liquid nitrogen, the tissue sample for taking 200mg to grind be placed in 2mL EP pipe
In, the beta -mercaptoethanol of 1mL polysaccharide polyphenol plant dedicated lysate and 50 μ L is added, whirlpool concussion immediately mixes, and is stored at room temperature
15min, after cracking it sufficiently, 4 DEG C, 12000r/min is centrifuged 10min；
S2, Aspirate supernatant are transferred in new 2mL EP pipe, operate on ice, the mixing of isometric chloroform and water-saturated phenol is added
Liquid (chloroform: water-saturated phenol=1:1), slight concussion mix, after being stored at room temperature layering 5min, 4 DEG C, and 12000r/min centrifugation
10min；
S3, Aspirate supernatant are transferred in new 2mL EP pipe, are operated on ice, and 200 μ L chloroforms are added, and slight concussion mixes, room
Warm stratification, then 4 DEG C, 12000r/min is centrifuged 10min.
S4, Aspirate supernatant are transferred in new 1.5mL EP pipe, are operated on ice, are added and isometric pre- are cooled to -20 DEG C
Dehydrated alcohol, -80 DEG C, stand 30min, after allowing RNA to be sufficiently precipitated, 4 DEG C, 12000r/min centrifugation 10min；
S5, supernatant is abandoned, the ethyl alcohol of 1mL 75% is added, after 5min is washed in mild concussion, 4 DEG C, 12000r/min is centrifuged 5min；
S6, abandoning supernatant, are added 1mL dehydrated alcohol, mildly shake after washing 5min, 4 DEG C, 12000r/min centrifugation 5min, in abandoning
Clearly, processing 20-30min is air-dried；
The mixed liquor that S7, addition are mixed by 10 μ LDNase and 70 μ L RDD buffers, mixes, is stored at room temperature about 15min；
S8,200 μ L RNase-free water are added, mix, adds 100 μ L chloroforms, mix gently, be stored at room temperature to layering；4
DEG C, 12000r/min is centrifuged 10min；
S9, Aspirate supernatant are transferred in new 1.5mL EP pipe, are operated on ice, and the anhydrous second that 1mL is cooled to -20 DEG C in advance is added
Alcohol, -80 DEG C, after standing 30min, 4 DEG C, 12000r/min is centrifuged 10min；
S10, supernatant is abandoned, 75% ethyl alcohol of 1mL is added, after 5min is washed in mild concussion, 4 DEG C, 12000r/min is centrifuged 5min；
S11, supernatant is abandoned, 1mL dehydrated alcohol is added, after 5min is washed in mild concussion, 4 DEG C, 12000r/min is centrifuged 5min；
S12, supernatant is abandoned, be air-dried at room temperature, 20-50 μ L RNase-free water is added and is dissolved to get total serum IgE, -80 DEG C are deposited
It puts.
The invention has the following advantages:
The protein that effectively prevents the degradation of carbohydrate and phenols to total serum IgE in plant, while can be mixed in fully erased RNA
And DNA.The extracted total serum IgE of this method can satisfy the research to Micro RNA and its target gene.
Detailed description of the invention
Fig. 1 is the RNA agarose gel figure for the polysaccharide polyphenol class plant extracted with Trizol method.
Fig. 2 is the extracted total serum IgE agarose gel figure of method using the embodiment of the present invention.
Specific embodiment
The present invention is described in detail combined with specific embodiments below.Following embodiment will be helpful to the technology of this field
Personnel further understand the present invention, but the invention is not limited in any way.It should be pointed out that the ordinary skill of this field
For personnel, without departing from the inventive concept of the premise, various modifications and improvements can be made.These belong to the present invention
Protection scope.
Embodiment
A method of polysaccharide polyphenol plant total serum IgE is extracted, is included the following steps:
S1, after taking appropriate tissue sample to be fully ground 3~5 times with liquid nitrogen, the tissue sample for taking about 200mg to grind is placed in 2mL EP
The dedicated lysate of 1mL polysaccharide polyphenol plant and 50 μ L beta -mercaptoethanols are added in Guan Zhong, and whirlpool concussion immediately mixes, and are stored at room temperature
15min, after cracking it sufficiently, 4 DEG C, 12000r/min is centrifuged 10min；
S2, Aspirate supernatant (about 900 μ L) are transferred in new 2mL EP pipe, are operated on ice, and isometric chloroform is added and water is full
With the mixed liquor (chloroform: water-saturated phenol=1:1) of phenol, slight concussion is mixed, after being stored at room temperature layering 5min, 4 DEG C, and 12000r/
Min is centrifuged 10min；
S3, Aspirate supernatant (about 800 μ L) are transferred in new 2mL EP pipe, are operated on ice, and 200 μ L chloroforms are added, slight to shake
Mixing is swung, layering is stored at room temperature, then 4 DEG C, 12000r/min is centrifuged 10min.
S4, Aspirate supernatant (about 700 μ l) are transferred in new 1.5mL EP pipe, are operated on ice, are added isometric pre-
It is cooled to -20 DEG C of dehydrated alcohol, -80 DEG C, stands 30min~2h, after allowing RNA to be sufficiently precipitated, 4 DEG C, 12000r/min centrifugation
10min；
S5, supernatant is abandoned, the ethyl alcohol of 1mL 75% is added, after about 5min is washed in mild concussion, 4 DEG C, 12000r/min is centrifuged 5min；
S6, supernatant is abandoned, 1mL dehydrated alcohol is added, after about 5min is washed in mild concussion, 4 DEG C, 12000r/min is centrifuged 5min, abandons
Supernatant air-dries processing about 20-30min；
The mixed liquor that S7, addition are mixed by 10 μ LDNase and 70 μ L RDD buffers, mixes, is stored at room temperature about 15min,
If ambient temperature is lower, the appropriate volume for increasing mixed liquor, this step is to remove the DNA in RNA；
S8,200 μ L RNase-free water are added, mix, adds 100 μ L chloroforms, mix gently, be stored at room temperature to layering；This
Step is to remove the DNase in RNA；
S9, Aspirate supernatant are transferred in new 1.5mL EP pipe, are operated on ice, and the anhydrous second that 1mL is cooled to -20 DEG C in advance is added
Alcohol, -80 DEG C, after standing 30min, 4 DEG C, 12000r/min is centrifuged 10min；
S10, supernatant is abandoned, 75% ethyl alcohol of 1mL is added, after about 5min is washed in mild concussion, 4 DEG C, 12000r/min is centrifuged 5min；
S11, supernatant is abandoned, 1mL dehydrated alcohol is added, after about 5min is washed in mild concussion, 4 DEG C, 12000r/min is centrifuged 5min；
S12, supernatant is abandoned, be air-dried at room temperature, 20-50 μ L RNase-free water is added and is dissolved to get total serum IgE, -80 DEG C are deposited
It puts.
Comparative example
1. tissue sample is directly placed into no RNase grinding body, a small amount of liquid nitrogen is added, grinds rapidly, repeatedly after 3-5 times,
50-100mg tissue is taken to be transferred to centrifuge tube, addition 1ml Trizol solution is sufficiently homogenized about with electric homogenizer and is needed 1-2 minutes,
It is placed at room temperature for 15min, cracks it sufficiently；
2.12000rpm is centrifuged 10min, abandons precipitating, and 200ul chloroform is added, and oscillation mixes 15 minutes, is placed at room temperature for 15min, and 4
DEG C, 12000g is centrifuged 15min；
3. drawing upper strata aqueous phase into another centrifuge tube, 0.5ml isopropanol is added, mixes, is placed at room temperature for 5-10min, 4 DEG C
12000g is centrifuged 10min, abandons supernatant, and RNA is sunken to tube bottom；
4. 75% ethyl alcohol of 1ml is added, centrifuge tube is mildly vibrated, suspend precipitating, and 4 DEG C of 8000g are centrifuged 5min, as far as possible abandoning supernatant,
5-10min is dried or be dried in vacuo to room temperature.Note: RNA sample not dried excessively, otherwise be difficult to dissolve.5. 50ul can be used
H2O, TE buffer or 0.5%SDS dissolution RNA sample, 55-60 DEG C, 5-10min.Note: H2O, TE or 0.5%SDS must be used
DEPC processing and high pressure.
In the RNA of extraction, if the band brightness of 18S is weaker than the band of 28S, illustrate extracted RNA than more complete,
Do not degrade.As shown in Figure 1, be the RNA agarose gel figure for the polysaccharide polyphenol class plant extracted with Trizol method, it can be with from figure
Find out that 18S band is bright in 28S band, RNA degradation is more severe.Moreover, remaining DNA and protein are more in RNA.Such as Fig. 2
It is shown, it is the extracted total serum IgE agarose gel figure of the present embodiment, it can be seen from the figure that extracted RNA there is no drop
Solution, and without the residual of DNA and protein.
Specific embodiments of the present invention are described above.It is to be appreciated that the invention is not limited to above-mentioned
Particular implementation, those skilled in the art can make a variety of changes or modify within the scope of the claims, this not shadow
Ring substantive content of the invention.In the absence of conflict, the feature in embodiments herein and embodiment can any phase
Mutually combination.
Claims (2)
1. a kind of method for extracting polysaccharide polyphenol plant total serum IgE, characterized by the following steps:
S1, after taking appropriate tissue sample to be fully ground 3~5 times with liquid nitrogen, the tissue sample for taking 200mg to grind be placed in 2mL EP pipe
In, the beta -mercaptoethanol of 1mL polysaccharide polyphenol plant dedicated lysate and 50 μ L is added, whirlpool concussion immediately mixes, and is stored at room temperature
15min, after cracking it sufficiently, 4 DEG C, 12000r/min is centrifuged 10min；
S2, Aspirate supernatant are transferred in new 2mL EP pipe, operate on ice, the mixing of isometric chloroform and water-saturated phenol is added
Liquid, slight concussion mix, and after being stored at room temperature layering 5min, 4 DEG C, 12000r/min is centrifuged 10min；
S3, Aspirate supernatant are transferred in new 2mL EP pipe, are operated on ice, and 200 μ L chloroforms are added, and slight concussion mixes, room
Warm stratification, then 4 DEG C, 12000r/min is centrifuged 10min.
S4, Aspirate supernatant are transferred in new 1.5mL EP pipe, are operated on ice, and isometric pre- nothing for being cooled to -20 DEG C is added
Water-ethanol, stands 30min by -80 DEG C, and after allowing RNA to be sufficiently precipitated, 4 DEG C, 12000r/min is centrifuged 10min；
S5, supernatant is abandoned, the ethyl alcohol of 1mL 75% is added, after 5min is washed in mild concussion, 4 DEG C, 12000r/min is centrifuged 5min；
S6, abandoning supernatant, are added 1mL dehydrated alcohol, mildly shake after washing 5min, 4 DEG C, 12000r/min centrifugation 5min, in abandoning
Clearly, processing 20-30min is air-dried；
The mixed liquor that S7, addition are mixed by 10 μ LDNase and 70 μ L RDD buffers, mixes, is stored at room temperature about 15min；
S8,200 μ L RNase-free water are added, mix, adds 100 μ L chloroforms, mix gently, be stored at room temperature to layering；4
DEG C, 12000r/min is centrifuged 10min；
S9, Aspirate supernatant are transferred in new 1.5mL EP pipe, are operated on ice, and the anhydrous second that 1mL is cooled to -20 DEG C in advance is added
Alcohol, -80 DEG C, after standing 30min, 4 DEG C, 12000r/min is centrifuged 10min；
S10, supernatant is abandoned, 75% ethyl alcohol of 1mL is added, after 5min is washed in mild concussion, 4 DEG C, 12000r/min is centrifuged 5min；
S11, supernatant is abandoned, 1mL dehydrated alcohol is added, after 5min is washed in mild concussion, 4 DEG C, 12000r/min is centrifuged 5min；
S12, supernatant is abandoned, be air-dried at room temperature, 20-50 μ L RNase-free water is added and is dissolved to get total serum IgE, -80 DEG C are deposited
It puts.
2. a kind of method for extracting polysaccharide polyphenol plant total serum IgE as described in claim 1, it is characterised in that: the step S2
In, the volume ratio of chloroform and water-saturated phenol is 1:1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910142165.5A CN109628443A (en) | 2019-02-26 | 2019-02-26 | A method of extracting polysaccharide polyphenol plant total serum IgE |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910142165.5A CN109628443A (en) | 2019-02-26 | 2019-02-26 | A method of extracting polysaccharide polyphenol plant total serum IgE |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109628443A true CN109628443A (en) | 2019-04-16 |
Family
ID=66065928
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910142165.5A Pending CN109628443A (en) | 2019-02-26 | 2019-02-26 | A method of extracting polysaccharide polyphenol plant total serum IgE |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109628443A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110195055A (en) * | 2019-07-02 | 2019-09-03 | 南宁维尔凯生物科技有限公司 | Polysaccharide polyphenol plant tissue method for extracting total RNA |
| CN110452906A (en) * | 2019-08-27 | 2019-11-15 | 上海美吉生物医药科技有限公司 | A method of rapidly and efficiently extracting lily petal total serum IgE |
| CN111073887A (en) * | 2020-01-23 | 2020-04-28 | 西南林业大学 | Extraction method and application of paphiopedilum high-quality total RNA |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104263720A (en) * | 2014-09-03 | 2015-01-07 | 北京林业大学 | Extraction method for total RNA of plant leaves |
-
2019
- 2019-02-26 CN CN201910142165.5A patent/CN109628443A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104263720A (en) * | 2014-09-03 | 2015-01-07 | 北京林业大学 | Extraction method for total RNA of plant leaves |
Non-Patent Citations (2)
| Title |
|---|
| 金征宇 等: "《基因与纳米探针-医学分子成像理论与实践》", 30 November 2017, 天津出版传媒集团 * |
| 黄璐琦 等: "《分子生药学》", 30 November 2008, 中国中医药出版社 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110195055A (en) * | 2019-07-02 | 2019-09-03 | 南宁维尔凯生物科技有限公司 | Polysaccharide polyphenol plant tissue method for extracting total RNA |
| CN110452906A (en) * | 2019-08-27 | 2019-11-15 | 上海美吉生物医药科技有限公司 | A method of rapidly and efficiently extracting lily petal total serum IgE |
| CN111073887A (en) * | 2020-01-23 | 2020-04-28 | 西南林业大学 | Extraction method and application of paphiopedilum high-quality total RNA |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109628443A (en) | A method of extracting polysaccharide polyphenol plant total serum IgE | |
| CN101638651B (en) | Method for extracting total RNA from plant tissue rich in polysaccharides and polyphenols and secondary metabolites | |
| Ghangal et al. | Isolation of good quality RNA from a medicinal plant seabuckthorn, rich in secondary metabolites | |
| Liu et al. | Direct DNA extraction method of an obligate parasitic fungus from infected plant tissue | |
| CN104313015A (en) | Method for extracting total RNA of polysaccharide and polyphenol plant tissues | |
| CN101845436B (en) | Method for simultaneously extracting total DNA and RNA from compost | |
| CN104263720B (en) | Plant leaf blade method for extracting total RNA | |
| CN104694531A (en) | Method for extracting total RNA from tea tissue | |
| CN103789197B (en) | A kind of test kit and extracting method thereof extracting Microrna | |
| CN113502286A (en) | Method for extracting DNA from plant tissue rich in secondary metabolites | |
| CN102876663B (en) | Reagent and method for rapidly extracting main RNA (ribonucleic acid) from liquid sample | |
| CN105002165A (en) | Method for improving extraction quality of total DNA of koji kaoliang spirit fermented grains through pretreatment | |
| CN104630209A (en) | Damage-free cold extraction method for genome DNA of small insects | |
| Wang et al. | Direct isolation of high-quality low molecular weight RNA of pear peel from the extraction mixture containing nucleic acid | |
| CN114395638B (en) | ATAC-seq method suitable for high starch fruits | |
| CN113797217B (en) | Use of anthocyanin derivatives for preparing medicine for treating cholesterol metabolism abnormality | |
| CN103421764B (en) | Kit for quickly extracting double-stranded RNA of mycovirus and application of kit | |
| CN105505920A (en) | Novel method for extracting recalcitrant plant tissue DNA | |
| CN104164421A (en) | Extraction method of grape peel RNA (ribonucleic acid) | |
| CN103409410B (en) | Method for extracting the total microbial genome in vinegar fermentation process | |
| CN110951724A (en) | Method for rapidly extracting nucleic acid DNA and/or RNA by Trizol | |
| CN110205319A (en) | A kind of simple and effective reagent and method for extracting plant pathogenic fungi genomic DNA | |
| CN106434640A (en) | Method for extracting RNA (ribonucleic acid) by aid of conventional reagents | |
| CN107475247A (en) | A kind of polyphenol polysaccharide vegetable RNA extracting method | |
| CN115058415B (en) | Rapid, high-quality and universal genome DNA extraction kit and DNA extraction method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190416 |
|
| RJ01 | Rejection of invention patent application after publication |