CN107012140A - It is a kind of to improve the method that RNA isolation kit extracts DNA from micro dry blade - Google Patents
It is a kind of to improve the method that RNA isolation kit extracts DNA from micro dry blade Download PDFInfo
- Publication number
- CN107012140A CN107012140A CN201710273257.8A CN201710273257A CN107012140A CN 107012140 A CN107012140 A CN 107012140A CN 201710273257 A CN201710273257 A CN 201710273257A CN 107012140 A CN107012140 A CN 107012140A
- Authority
- CN
- China
- Prior art keywords
- adsorption column
- added
- dna
- dry
- rna isolation
- 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
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
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)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The method that RNA isolation kit extracts DNA from micro dry blade is improved the invention discloses a kind of, is concretely comprised the following steps：Take 20mg to dry extra large leaf tissue to add in freeze grinding centrifuge tube, grinding obtains dry powder；Dry powder is added 500ulLP1 and 6ulRNase and is vortexed and is shaken；Add 130ul buffer solutions LP2；Supernatant is transferred in new centrifuge tube；The buffer solution LP3 of 1.5 times of addition；Solution and precipitation are all added in same adsorption column CB3, adsorption column is put into collecting pipe, 30sec is centrifuged, outwells waste liquid；Rinsing liquid PW is added into adsorption column, centrifugation outwells waste liquid, repeats this step once；Adsorption column is put back in collecting pipe, is centrifuged, is outwelled waste liquid, adsorption column is put into new centrifuge tube, thoroughly dry the remaining rinsing liquid in adsorption column, obtain adsorbed film；Nuclease-free water is vacantly added to adsorbed film middle part, 5min is centrifuged, takes out and abandon adsorption column；To DNA concentration and purity detecting, 20 DEG C of preservation samples.The present invention has the advantages that extraction convenience, quality are good.
Description
Technical field
The present invention relates to DNA of plants preparing technical field, specifically a kind of RNA isolation kit of improveing is from micro dried leaf
The method that DNA is extracted in piece.
Background technology
Nucleic acid is the main compound for constituting basic genetic material in life entity, is ground from nucleic acid molecules system horizontal is deep
It is the basis for recognizing various biological phenomenas to study carefully.The extraction of DNA of plants be mainly used in construction of gene library, PCR analysis and
Southern hybridization etc., therefore high-quality DNA acquisition has important meaning for functional genomics and genetics research
Justice.At present, in plant extract DNA research, a variety of methods have been developed, successfully from plant leaf blade, callus, group
DNA is extracted in the histoorgans such as training seedling, fruit, first skin zone.Using fresh or freezing material more than these methods, but for open country
Outside, especially remote sampling, carries curling stone or liquid nitrogen is very inconvenient, and it is special that this needs makees some before DNA is extracted to sample
Different processing, studies have found that can be used for the extraction of plant genome DNA by the piece after silica dehydrator, no matter but tender
The blade of blade or maturation, though the rapid dehydration of blade, also contains substantial amounts of polysaccharide, polyphenol, albumen in plant leaf blade tissue
With the Secondary metabolites such as esters and the DNA of degraded, this to extract DNA often occur yield poorly, of poor quality, easy drop
Solution, have impact on DNA mass and purity, it is impossible to which, by digestion with restriction enzyme, the serious template that even cannot function as enters performing PCR expansion
Increase, being extracted with fresh leaf material has very big difference.
Ecological construction, environmental protection are increasingly subject to everybody concern, and plant is important Natural Resources Environment key element, for
Maintain the ecological balance and important role of developing the economy, however as the development of science and technology, our life increasingly improves can
It is that our earth but receives harm, the forest reserves are by serious destruction, many precious going to wreck property of plant resources
Strike, therefore, DNA of plants extraction research to the in-situ protection of plant, germ plasm resource Collection and conservation and introduces a collection cultivation work
It is particularly important.
At present, the report overwhelming majority on Method of Plant DNA Extraction is changed on the basis of SDS methods and CTAB methods
It is good, but the operating procedure ground one by one to sample with grinding rod or glass bar can not be all broken away from, and extraction step is cumbersome time-consuming, passes
The CTAB methods of system are current most widely used DNA extraction methods, but this method is when extracting plant drying blade, residual
Substantial amounts of albumen glucide, has influence on follow-up PCR amplifications, analysis of genetic polymorphisms research, so far, yet there are no for plant
Dry the research report that high quality DNA is extracted in blade.
The content of the invention
The invention aims to solve, extraction complexity of the prior art is cumbersome, can not extract lacking for high quality DNA
Fall into and extract DNA method from micro dry blade to solve the above problems there is provided a kind of improvement RNA isolation kit.
The method that RNA isolation kit extracts DNA from micro dry blade is improved the invention discloses a kind of, is concretely comprised the following steps：
(1), take 20mg to dry extra large leaf tissue to be added in 2.0ml freeze grinding centrifuge tubes, and be put into 1 steel that sterilized
Pearl, liquid nitrogen frozen 3-5min, centrifuge tube is put into beveller and grinds 8-12min, obtains dry powder；
(2), the dry powder for obtaining step (1) adds 500ulLP1 and 6ulRNase, and be vortexed concussion 1min, and room temperature is placed
15min, during which turns upside down 5-10 times；
(3), into step (2), product adds 130ul buffer solution LP2, and be vortexed concussion 1min；
(4), the centrifuge tube in step (3) is placed in a centrifuge, 12000rpm (~13400*g) centrifugation 5min will be upper
Clear liquid is transferred in new 1.5ml centrifuge tubes；
(5) 1.5 times of buffer solution LP3 is added in the supernatant, obtained to step (4), fully shaking mixes 15-30sec；
(6) solution and precipitation for, obtaining step (5) are all added in same adsorption column CB3, and adsorption column is put into receipts
In collector, 12000rpm (~13400*g) centrifugation 30sec outwell waste liquid, again put back to adsorption column CB3 in collecting pipe；
(7) 600ul rinsing liquids PW, 12000rpm (~13400*g) centrifugation 30sec, are added into adsorption column, is outwelled useless
Liquid, repeats this step once；
(8), adsorption column is put back in collecting pipe, 12000rpm (~13400*g) centrifugation 3min outwell waste liquid, will adsorbed
Post is put into new 1.5ml centrifuge tubes, and room temperature places 5-10min, thoroughly dries the remaining rinsing liquid in adsorption column, is adsorbed
Film；
(9) 50ul nuclease-free waters vacantly, are added to adsorbed film middle part, room temperature places 5min, 12000rpm (~
5min 13400*g) is centrifuged, takes out and abandons adsorption column；
(10), to DNA concentration and purity detecting, -20 DEG C of preservation samples.
Preferably, in described step (1), it is necessary to the first precooling of beveller before centrifuge tube is put into beveller；
Preferably, in described step (1), before it will dry extra large leaf tissue addition freeze grinding centrifuge tube, first
80% ethanol configured using sterilized water is rinsed, after drying, and extra large leaf tissue is shredded；
Preferably, in described step (2), described RNase concentration is 10mg/ml；
Preferably, in described step (4), should not during supernatant to be transferred to new 1.5ml centrifuge tubes
Encounter precipitation；
Preferably, in described step (9), when nuclease-free water is added into adsorbed film middle part, it is necessary to first will
Nuclease-free water is preheated to 65 DEG C.
The present invention has advantages below compared with prior art：
According to the present invention, on the basis of conventional reagents box and polishing, step is adjusted flexibly, makes to dry blade quickly high
The grind into powder extracting DNA of effect, can extract high-quality genomic DNA, extraction comparison is convenient from micro dry blade.
Brief description of the drawings
Fig. 1 is the agarose gel electrophoresis figure that embodiments of the invention extract DNA with CTAB methods；
Fig. 2 is that improvement RNA isolation kit extracting DNA, 2ul dilute DNA PCR electrophoretograms after 10 times of dilutions.
Embodiment
Embodiments of the invention are elaborated below, the present embodiment is carried out lower premised on technical solution of the present invention
Implement, give detailed embodiment and specific operating process, but protection scope of the present invention is not limited to following implementations
Example.
The method that RNA isolation kit extracts DNA from micro dry blade is improved the invention discloses a kind of, is concretely comprised the following steps：
(1), take 20mg to dry extra large leaf tissue to be added in 2.0ml freeze grinding centrifuge tubes, and be put into 1 steel that sterilized
Pearl, liquid nitrogen frozen 3-5min, centrifuge tube is put into beveller and grinds 8-12min, obtains dry powder；
(2), the dry powder for obtaining step (1) adds 500ulLP1 and 6ulRNase, and be vortexed concussion 1min, and room temperature is placed
15min, during which turns upside down 5-10 times；
(3), into step (2), product adds 130ul buffer solution LP2, and be vortexed concussion 1min；
(4), the centrifuge tube in step (3) is placed in a centrifuge, 12000rpm (~13400*g) centrifugation 5min will be upper
Clear liquid is transferred in new 1.5ml centrifuge tubes；
(5) 1.5 times of buffer solution LP3 is added in the supernatant, obtained to step (4), fully shaking mixes 15-30sec；
(6) solution and precipitation for, obtaining step (5) are all added in same adsorption column CB3, and adsorption column is put into receipts
In collector, 12000rpm (~13400*g) centrifugation 30sec outwell waste liquid, again put back to adsorption column CB3 in collecting pipe；
(7) 600ul rinsing liquids PW, 12000rpm (~13400*g) centrifugation 30sec, are added into adsorption column, is outwelled useless
Liquid, repeats this step once；
(8), adsorption column is put back in collecting pipe, 12000rpm (~13400*g) centrifugation 3min outwell waste liquid, will adsorbed
Post is put into new 1.5ml centrifuge tubes, and room temperature places 5-10min, thoroughly dries the remaining rinsing liquid in adsorption column, is adsorbed
Film；
(9) 50ul nuclease-free waters vacantly, are added to adsorbed film middle part, room temperature places 5min, 12000rpm (~
5min 13400*g) is centrifuged, takes out and abandons adsorption column；
(10), to DNA concentration and purity detecting, -20 DEG C of preservation samples.
Preferably, in described step (1), it is necessary to the first precooling of beveller before centrifuge tube is put into beveller；
Preferably, in described step (1), before it will dry extra large leaf tissue addition freeze grinding centrifuge tube, first
80% ethanol configured using sterilized water is rinsed, after drying, and extra large leaf tissue is shredded；
Preferably, in described step (2), described RNase concentration is 10mg/ml；
Preferably, in described step (4), should not during supernatant to be transferred to new 1.5ml centrifuge tubes
Encounter precipitation；
Preferably, in described step (9), when nuclease-free water is added into adsorbed film middle part, it is necessary to first will
Nuclease-free water is preheated to 65 DEG C.
Embodiment 1
The method that RNA isolation kit extracts DNA from micro dry blade is improved the invention discloses a kind of, is concretely comprised the following steps：
(1) different growing stage of kindred plant, is taken to dry extra large leaf tissue 20mg, 80% second configured using sterilized water
Alcohol is rinsed, and will be dried blade and be shredded and be transferred in 2.0ml freeze grinding centrifuge tubes, and is put into 1 steel ball that sterilized,
Liquid nitrogen frozen 4min, and centrifuge tube is put into Liquid nitrogen precooler beveller grinds 10min, obtain dry powder；
(2), the dry powder for obtaining step (1) adds 500ulLP1 and 6ulRNase, and be vortexed concussion 1min, and room temperature is placed
15min, during which turns upside down 7 times；
(3), into step (2), product adds 130ul buffer solution LP2, and be vortexed concussion 1min；
(4), the centrifuge tube in step (3) is placed in a centrifuge, 12000rpm (~13400*g) centrifugation 5min will be upper
Clear liquid is transferred in new 1.5ml centrifuge tubes；
(5) 1.5 times of buffer solution LP3 is added in the supernatant, obtained to step (4), fully shaking mixes 20sec；
(6) solution and precipitation for, obtaining step (5) are all added in same adsorption column CB3, and adsorption column is put into receipts
In collector, 12000rpm (~13400*g) centrifugation 30sec outwell waste liquid, again put back to adsorption column CB3 in collecting pipe；
(7) 600ul rinsing liquids PW, 12000rpm (~13400*g) centrifugation 30sec, are added into adsorption column, is outwelled useless
Liquid, repeats this step once；
(8), adsorption column is put back in collecting pipe, 12000rpm (~13400*g) centrifugation 3min outwell waste liquid, will adsorbed
Post is put into new 1.5ml centrifuge tubes, and room temperature places 7min, thoroughly dries the remaining rinsing liquid in adsorption column, obtains adsorbed film；
(9) 50ul nuclease-free waters vacantly, are added to adsorbed film middle part, room temperature places 5min, 12000rpm (~
5min 13400*g) is centrifuged, takes out and abandons adsorption column；
(10), to DNA concentration and purity detecting, -20 DEG C of preservation samples.
Preferably, in described step (1), it is necessary to the first precooling of beveller before centrifuge tube is put into beveller；
Preferably, in described step (2), described RNase concentration is 10mg/ml；
Preferably, in described step (4), should not during supernatant to be transferred to new 1.5ml centrifuge tubes
Encounter precipitation；
Preferably, in described step (9), when nuclease-free water is added into adsorbed film middle part, it is necessary to first will
Nuclease-free water is preheated to 65 DEG C.
The concentration and purity for extracting DNA are determined using NanoDrop, agarose gel electrophoresis Detection and Extraction DNA's is complete
Property, the A260/A280 of sample can be seen that contrast using tradition 1.8 or so from NanoDrop and agarose electrophoresis after measured
CTAB methods extract dry leaf DNA, although DNA can be obtained, DNA purity and concentration is very low, poor quality, relatively
, it can be extracted on the premise of same sample amount and obtain purity and concentration height, the good gene of integrality using the method for embodiment
Group DNA, efficient amplification, the rare precious sample of the comparison that is particularly suitable for use in are carried out in follow-up PCR as template.
General principle, principal character and the advantages of the present invention of the present invention has been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and that described in above-described embodiment and specification is the present invention
Principle, various changes and modifications of the present invention are possible without departing from the spirit and scope of the present invention, these change and
Improvement is both fallen within the range of claimed invention.The protection domain of application claims by appended claims and its
Equivalent is defined.
Claims (6)
1. a kind of improve the method that RNA isolation kit extracts DNA from micro dry blade, it is characterised in that：Concretely comprise the following steps：
(1), take 20mg to dry extra large leaf tissue to be added in 2.0ml freeze grinding centrifuge tubes, and be put into 1 steel ball that sterilized,
Liquid nitrogen frozen 3-5min, centrifuge tube is put into beveller and grinds 8-12min, obtains dry powder；
(2), the dry powder for obtaining step (1) adds 500ulLP1 and 6ulRNase, and be vortexed concussion 1min, and room temperature places 15min,
Period turns upside down 5-10 times；
(3), into step (2), product adds 130ul buffer solution LP2, and be vortexed concussion 1min；
(4), the centrifuge tube in step (3) is placed in a centrifuge, 12000rpm (~13400*g) centrifugation 5min, by supernatant
It is transferred in new 1.5ml centrifuge tubes；
(5) 1.5 times of buffer solution LP3 is added in the supernatant, obtained to step (4), fully shaking mixes 15-30sec；
(6) solution and precipitation for, obtaining step (5) are all added in same adsorption column CB3, and adsorption column is put into collecting pipe
In, 12000rpm (~13400*g) centrifugation 30sec outwell waste liquid, again put back to adsorption column CB3 in collecting pipe；
(7) 600ul rinsing liquids PW, 12000rpm (~13400*g) centrifugation 30sec, are added into adsorption column, waste liquid is outwelled, weight
This multiple step is once；
(8), adsorption column is put back in collecting pipe, 12000rpm (~13400*g) centrifugation 3min outwell waste liquid, adsorption column is put
Enter in new 1.5ml centrifuge tubes, room temperature places 5-10min, thoroughly dries the remaining rinsing liquid in adsorption column, obtains adsorbed film；
(9) 50ul nuclease-free waters vacantly, are added to adsorbed film middle part, room temperature places 5min, 12000rpm (~13400*
G) 5min is centrifuged, takes out and abandons adsorption column；
(10), to DNA concentration and purity detecting, -20 DEG C of preservation samples.
2. the method that a kind of improvement RNA isolation kit according to claim 1 extracts DNA from micro dry blade, its feature
It is：In described step (1), it is necessary to the first precooling of beveller before centrifuge tube is put into beveller.
3. the method that a kind of improvement RNA isolation kit according to claim 1 or 2 extracts DNA from micro dry blade, its
It is characterised by：In described step (1), before it will dry extra large leaf tissue addition freeze grinding centrifuge tube, first using sterile
80% ethanol of water configuration is rinsed, and after drying, extra large leaf tissue is shredded.
4. the method that a kind of improvement RNA isolation kit according to claim 1 extracts DNA from micro dry blade, its feature
It is：In described step (2), described RNase concentration is 10mg/ml.
5. the method that a kind of improvement RNA isolation kit according to claim 1 extracts DNA from micro dry blade, its feature
It is：In described step (4), during supernatant to be transferred to new 1.5ml centrifuge tubes, precipitation should not be encountered.
6. the method that a kind of improvement RNA isolation kit according to claim 1 extracts DNA from micro dry blade, its feature
It is：In described step (9), when nuclease-free water is added into adsorbed film middle part, it is necessary to first that nuclease-free water is pre-
Heat is to 65 DEG C.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710273257.8A CN107012140A (en) | 2017-04-25 | 2017-04-25 | It is a kind of to improve the method that RNA isolation kit extracts DNA from micro dry blade |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710273257.8A CN107012140A (en) | 2017-04-25 | 2017-04-25 | It is a kind of to improve the method that RNA isolation kit extracts DNA from micro dry blade |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107012140A true CN107012140A (en) | 2017-08-04 |
Family
ID=59447031
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710273257.8A Pending CN107012140A (en) | 2017-04-25 | 2017-04-25 | It is a kind of to improve the method that RNA isolation kit extracts DNA from micro dry blade |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107012140A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109652408A (en) * | 2019-02-28 | 2019-04-19 | 广东美立康生物科技有限公司 | The method for saving rapidly extracting high concentration DNA in sample from micro alcoholic solution |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104004838A (en) * | 2014-05-21 | 2014-08-27 | 全国畜牧总站 | Sudangrass variety authenticity detection method and special primers |
-
2017
- 2017-04-25 CN CN201710273257.8A patent/CN107012140A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104004838A (en) * | 2014-05-21 | 2014-08-27 | 全国畜牧总站 | Sudangrass variety authenticity detection method and special primers |
Non-Patent Citations (1)
| Title |
|---|
| Y.X. LI等: "Identification of a locus characteristic of male individuals of buffalo grass [Buchloe dactyloides (Nutt.) Engelm.] by using an RAPD marker", 《GENETICS AND MOLECULAR RESEARCH》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109652408A (en) * | 2019-02-28 | 2019-04-19 | 广东美立康生物科技有限公司 | The method for saving rapidly extracting high concentration DNA in sample from micro alcoholic solution |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Richards et al. | Preparation of genomic DNA from plant tissue | |
| Zhu et al. | Isolation of genomic DNAs from plants, fungi and bacteria using benzyl chloride. | |
| Opel et al. | The application of miniplex primer sets in the analysis of degraded DNA from human skeletal remains | |
| CN104178480B (en) | Using the kit and method of DNA adsorption column rapid extraction DNA of plants | |
| Marsal et al. | A fast, efficient method for extracting DNA from leaves, stems, and seeds of Vitis vinifera L. | |
| CN110567951A (en) | Apple stem groove virus visual detection system based on CRISPR-Cas12a technology and detection method thereof | |
| CN102286467A (en) | Method for extracting microbial total RNA in forest soil and litter | |
| CN101709298A (en) | Soil DNA extracting method for evaluating diversity of microbial community of plant root system | |
| CN104561332A (en) | SSR molecular marker for identifying aspen gender and application of SSR molecular marker | |
| CN105859831A (en) | Apple protein extraction method and related protein extracting solution | |
| CN103320425B (en) | Rapid extraction method of shellfish deoxyribonucleic acid (DNA) for large-scale and high-throughput genotyping | |
| CN104531679A (en) | Method for extracting DNA from dry apricot leaf | |
| CN101845436A (en) | Method for simultaneously extracting total DNA and RNA from compost | |
| CN105648106B (en) | A kind of Exserohilum turcicum molecular detection primer and rapid detection method | |
| CN107012140A (en) | It is a kind of to improve the method that RNA isolation kit extracts DNA from micro dry blade | |
| CN101899435A (en) | High-throughput extraction method for sugarcane leaf genome by using ball mill | |
| CN110951917B (en) | RNA-RP RT-PCR method for rapidly detecting melon viruses | |
| CN104694530A (en) | Extraction method of wheat genome DNA | |
| Lahuf et al. | A simple, rapid, safe and low-cost method to extract DNA from phytopathogenic fungi. | |
| CN105063016A (en) | Method for extracting microorganism total DNA from yellow rice wine wheat koji | |
| CN105255857A (en) | Camellia sinensis DNA extraction method | |
| CN104164423B (en) | Kit and method for rapidly extracting plant RNA by utilizing RNA adsorption column | |
| CN105505916A (en) | Method for extracting high-quality genome DNA from dry suriana maritime leaf and kit thereof | |
| JPWO2005073377A1 (en) | Method for recovering DNA from environmental samples | |
| CN103421764B (en) | Kit for quickly extracting double-stranded RNA of mycovirus and application of kit |
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: 20170804 |
|
| RJ01 | Rejection of invention patent application after publication |