CN104862305A - Animal tissue genomic DNA and RNA rapid extraction kit, extraction method and application - Google Patents
Animal tissue genomic DNA and RNA rapid extraction kit, extraction method and application Download PDFInfo
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- CN104862305A CN104862305A CN201510333776.XA CN201510333776A CN104862305A CN 104862305 A CN104862305 A CN 104862305A CN 201510333776 A CN201510333776 A CN 201510333776A CN 104862305 A CN104862305 A CN 104862305A
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Abstract
The invention relates to an animal tissue genomic DNA and RNA rapid extraction kit which includes a lysate, a wash solution 1, a wash solution 2, absolute ethyl alcohol and a diethylpyrocarbonate aqueous solution. The extraction method includes the following steps: the solid animal tissue is firstly grinded during extraction, and the liquid animal tissue is extracted directly; after the sample is split, the genomic DNA and RNA are adsorbed on DNA and RNA adsorption columns; then the diethylpyrocarbonate aqueous solution is used to wash to obtain the whole genome DNA and RNA. The kit can be used for simultaneous extraction and purification of genomic DNA and RNA of animal tissue; with the method, the whole extraction process only needs 5 minutes, the extraction efficiency is high, the detection sensitivity is high, and reliable experimental basis is provided for early diagnosis of various types of DNA and RNA bacteria or viral pathogens infection and early diagnosis; the animal tissue genomic DNA and RNA rapid extraction kit and the extraction method are applicable to all research institutions, clinical pathogen detection, molecular genetics research, clinical genetic diagnosis, and animal disease diagnosis and disease control centers, and have broad market prospects and greater economic benefits.
Description
Technical field
The present invention relates to a kind of for animal tissues's genomic dna and RNA rapid extraction test kit and extracting method and application, belong to biological technical field.
Background technology
In molecular biology research, extraction purification DNA and RNA is the first step of molecular test, all needs extraction purification DNA and RNA in various clinical detection or scientific research.At present, the test kit of existing multiple PCR-based, RT-PCR technology detection DNA and RNA is applied in clinical diagnosis and detection both at home and abroad, the DNA that these test kits provide and RNA extraction method mainly phenol chloroform extraction method.Phenol, chloroform are the denaturing agents of DNA and RNA leaching process, and extracting can remove protein, salt and impurity repeatedly.But phenol and chloroform have larger toxicity to human body, volatility is comparatively strong, and whole leaching process complex steps, consuming time longer, often easily cause the degraded of DNA and RNA, and increase energy consumption and experimentation cost, often disadvantageous effect is caused to downstream experiment.
Full-length genome is that cell or tissue comprises encoding sequence and non-coding sequence in interior full gene information, and nearly all life-information all can find from full-length genome, comprises the gene's expression and control relevant to Animal diseases.At present, pathogeny detection and molecular diagnosis are technology common in clinical detection, and the purity that its susceptibility, specificity and DNA and RNA extract and concentration have much relations, directly determine detected result.Detect with molecular diagnosis in Animal diseases, highly sensitive and specific molecular diagnosis progressively replace Serological testing.Thus, obtaining the genomic dna of high purity and high density and RNA is the first step in animal pathogen scientific research and clinical detection, which determining the specificity that amplification combines and diagnoses.
The extraction of DNA and RNA is a kind of technology conventional in molecular biology research, has multiple DNA and RNA extraction method in the market.Containing a large amount of protein, salt and impurity in animal tissues, the character of its character and nucleic acid is closely similar, be difficult to isolate from DNA and RNA extracted, in DNA and the RNA leaching process of animal tissues, be easy to and DNA and RNA coprecipitation, and be difficult to be separated, often cause DNA and the RNA purity of extraction and concentration inadequate, have impact on follow-up amplification and order-checking.
For reducing or removing protein, salt and the impurity in animal tissues, in existing DNA and RNA leaching process, usually there is pollution, and complex steps, the leaching process time is longer.As extracting method such as sodium perchlorate method, SDS method and Wyler's processs, but these methods need sample size larger, and the organism such as phenol are easily residual in DNA and RNA solution, particularly amplification procedure can be disturbed in follow-up PCR, RT-PCR amplification procedure, affect amplification, the application of the method is subject to certain restrictions.
The method adopted in the market is: (1) adopts chloroform, phenol extraction method removes protein, salt and impurity.(2) first step extracting DNA and RNA is lysate sample, and disclosed method is for mostly being water-bath cracking 15 minutes to 1 hour in the market, and pyrolysis time is longer, adds testing cost.
For animal tissues, adopt molecular biology method study its diagnosis, first to carry out be exactly bacterium or virus in sample DNA and RNA extract.DNA and RNA of different sorts bacterium and virus can be obtained, and genomic integrity, purity and concentration, be from animal tissues, extract the primary goal of DNA and RNA for animal pathogen diagnosis research.Therefore, need one to be suitable for use in the super rapid extraction test kit of Animal genome DNA and RNA, the demand of current rapid molecular diagnosis could be met, ensure the sound development of livestock industry.
Summary of the invention
Object of the present invention: be to provide a kind of for animal tissues's genomic dna and RNA rapid extraction test kit and application thereof, present method is easy and simple to handle, saving of work and time, the demand of the more and DNA rapid extraction of sample, RNA can be met, for the sound development of animal pathogen quick diagnosis and aquaculture provides technical support and theoretical basis.
The object of the invention is to be achieved through the following technical solutions:
A kind of rapid extraction test kit for animal tissues's genomic dna and RNA, comprise following reagent: lysate, washing fluid 1, washing fluid 2, dehydrated alcohol and weight ratio are the diethylpyrocarbonate aqueous solution of 0.5 ~ 1.5%, and wherein the composition of lysate, washing fluid 1, washing fluid 2 is respectively:
(1) lysate: EDTA 30 ~ 50 mM, the Triton X-100 of weight ratio 1 ~ 3%, lsothiocyanates 1 ~ 3 M, 0.5 ~ 0.9 M sodium-chlor;
(2) washing fluid 1:5 ~ 15 mM EDTA, the ethanol of volume ratio 70%, pH 7.0;
(3) washing fluid 2: containing 65 ~ 85 mM Tris-HCl, the ethanol of volume ratio 80%, pH 7.0.
The described rapid extraction test kit for animal tissues's genomic dna and RNA, wherein: the concentration of the diethylpyrocarbonate aqueous solution is 1%;
Lysate: EDTA 40 mM, the Triton X-100 of weight ratio 2%, lsothiocyanates 2 M, 0.7 M sodium-chlor;
Washing fluid 1:10 mM EDTA, the ethanol of volume ratio 70%, pH 7.0;
Washing fluid 2: containing 75 mM Tris-HCl, the ethanol of volume ratio 80%, pH 7.0.
Utilize described DNA and RNA rapid extraction test kit to extract the genomic method of animal tissues, comprise the following steps:
(1) animal tissues of solid state is first carried out milled processed, liquid animal tissues is directly used in extraction;
(1) get liquid animal tissues 20 μ l ~ 100 μ l, or get solid state animal tissues 20mg ~ 100 mg after grinding, add 500 μ l lysates, 70 DEG C of water-bath 3-5 min, 12000 rpm 30 S, isolate supernatant liquor;
(2) proceed in centrifuge tube by supernatant liquor, add 500 μ l dehydrated alcohols, gradation forwards on DNA and RNA purification column, centrifugal 30 S of 12000 rpm; DNA and RNA----slightly carried, outwells the waste liquid in collection tube;
(3) purification column is rinsed with 500 μ l washing fluids 1----, centrifugal 30 S of 12000 rpm, outwell the waste liquid in collection tube;
(4) rinse purification column with 600 μ l washing fluids 2, centrifugal 30 S of 12000 rpm, outwell the waste liquid in collection tube;
(5) rinse purification column with 500 μ l washing fluids 2, centrifugal 30 S of 12000 rpm, outwell the waste liquid in collection tube;
(6) 12000 centrifugal 30 S again, obtain DNA and RNA----;
(7) forward DNA and RNA purification column to another centrifuge tube, add the 50 μ l diethylpyrocarbonate aqueous solution, 12000 centrifugal 30 S, can obtain DNA and RNA of highly purified virus and tissue gene group, save backup in-20 DEG C.
The animal tissues of described solid state is heart, liver, lungs, spleen or kidney; Described liquid animal tissues is whole blood, blood plasma, serum or animal secretions.
Described test kit is for the application in animal tissues's genomic dna and RNA rapid extraction.
positive beneficial effect of the present invention:
Animal tissues of the present invention genomic dna and RNA rapid extraction test kit, can DNA and RNA of rapid extraction animal tissues, for the quick diagnosis of animal pathogen, may be used for again molecule genetics research, clinical gene diagnosis and animal diseases diagnosis.Test kit of the present invention also may be used for other similar tissue DNAs and the extraction of RNA and the diagnosis of pathogenic bacteria.
Test kit DNA of the present invention and RNA extraction efficiency is high, detection sensitivity is high, infects for early diagnosis all kinds of DNA and RNA bacterium or viral pathogen and early diagnosis provides reliable experimental basis.
Sample in test kit of the present invention is after cracking, genomic dna and RNA are attracted on DNA and RNA adsorption column respectively, then DNA and RNA that can obtain full-length genome is rinsed with DEPC water, in leaching process, DNA with RNA agents useful for same is identical, and difference is exactly DNA purification column and RNA purification column.Easy and simple to handle, saving of work and time, extraction rate is ultrafast, and whole leaching process only needs 5 minutes, can meet the demand of the more and DNA rapid extraction of sample and RNA, for the sound development of animal pathogen quick diagnosis and aquaculture provides technical support and theoretical basis.
Test kit of the present invention is for extracting animal tissues's (as heart, liver, lungs, spleen, kidney), the genomic dna of whole blood, blood plasma, serum and animal secretions (emulsion, nose liquid) and RNA, and this genomic dna and RNA can be used for RT-PCR reaction, RAPD, AFLP, RFLP and viral genome amplification.
The present invention is applicable to the clinical Pathogen test of R&D institution, molecule genetics research, clinical gene diagnosis and animal diseases diagnosis and Center for Disease Control, has wide market outlook and good economic benefit.
Embodiment
Embodiment 1: a kind of be used for animal tissues's genomic dna and RNA rapid extraction test kit and extracting method
(1) kit reagent: comprise the diethylpyrocarbonate aqueous solution that lysate, washing fluid 1, washing fluid 2 and dehydrated alcohol and concentration are 1%;
Lysate: EDTA 40 mM(mmol/L), the Triton X-100 of weight ratio 2%, lsothiocyanates 2 M(mol/L), 0.7 M sodium-chlor;
Washing fluid 1:10 mM EDTA, the ethanol of volume ratio 70%, pH 7.0;
Washing fluid 2: containing 75 mM Tris-HCl, the ethanol of volume ratio 80%, pH 7.0.
Extract animal tissues as heart, liver, lungs, spleen, kidney etc. with this test kit, these animal tissuess need first to grind to form pasty state, then extract, and extraction step is as follows:
(1) get the animal tissues 20mg after grinding, add 500 μ l lysates, 70 DEG C of water-bath 2 min, 12000 rpm 30 S, isolate supernatant liquor;
(2) proceed in centrifuge tube by supernatant liquor, add 500 μ l dehydrated alcohols, gradation forwards on DNA and RNA purification column, centrifugal 30 S of 12000 rpm; DNA and RNA----slightly carried, outwells the waste liquid in collection tube;
(3) rinse purification column with 500 μ l washing fluids 1, centrifugal 30 S of 12000 rpm, outwell the waste liquid in collection tube;
(4) rinse purification column with 600 μ l washing fluids 2, centrifugal 30 S of 12000 rpm, outwell the waste liquid in collection tube;
(5) rinse purification column with 500 μ l washing fluids 2, centrifugal 30 S of 12000 rpm, outwell the waste liquid in collection tube;
(6) 12000 centrifugal 30 S again, obtain genomic dna and RNA;
(7) forward DNA and RNA purification column to another centrifuge tube, add the DEPC aqueous solution of 50 μ l 1%, centrifugal 30 S of 12000 rpm, can obtain DNA and RNA of highly purified virus and tissue gene group, save backup in-20 DEG C.
embodiment 2: a kind of be used for animal tissues's genomic dna and RNA rapid extraction test kit and extracting method, substantially identical with example 1, difference is:
Kit reagent: comprise lysate, washing fluid 1, washing fluid 2 and dehydrated alcohol and 0.5%(weight ratio) the diethylpyrocarbonate aqueous solution.
Each agent formulations:
(1) lysate: EDTA 30 mM, the Triton X-100 of weight ratio 1%, the lsothiocyanates of 1M, the sodium-chlor of 0.5 M;
(2) EDTA of washing fluid 1:5 mM, the ethanol of volume ratio 70%, pH 7.0;
(3) washing fluid 2: containing 65mM Tris-HCl, the ethanol of volume ratio 80%, pH 7.0.
embodiment 3: a kind of be used for animal tissues's genomic dna and RNA rapid extraction test kit and extracting method
(1) test kit comprises lysate, washing fluid 1, washing fluid 2, dehydrated alcohol and 0.5 ~ 1.5%(weight ratio) the diethylpyrocarbonate aqueous solution.
Each agent formulations:
Lysate: EDTA 40 mM, 2% Triton X-100, lsothiocyanates 2 M, 0.7 M sodium-chlor;
Washing fluid 1:10 mM EDTA, the ethanol of volume ratio 70%, pH 7.0;
Washing fluid 2: containing 75 mM Tris-HCl, the ethanol of volume ratio 80%, pH 7.0.
Genomic dna and RNA by following Program extraction animal tissues (heart, liver, lungs, spleen, kidney), whole blood, blood plasma, serum, animal secretions (emulsion, nose liquid):
(1) get above animal tissues 50 μ l, add 500 μ l lysates, 70 DEG C of water-bath 2 min, 12000 rpm 30 S, isolate supernatant liquor;
(2) proceed in centrifuge tube by supernatant liquor, add 500 μ l dehydrated alcohols, gradation forwards on DNA and RNA purification column, centrifugal 30 S of 12000 rpm; DNA and RNA----slightly carried, outwells the waste liquid in collection tube;
(3) rinse purification column with 500 μ l washing fluids 1, centrifugal 30 S of 12000 rpm, outwell the waste liquid in collection tube;
(4) rinse purification column with 600 μ l washing fluids 2, centrifugal 30 S of 12000 rpm, outwell the waste liquid in collection tube;
(5) rinse purification column with 500 μ l washing fluids 2, centrifugal 30 S of 12000 rpm, outwell the waste liquid in collection tube;
(6) centrifugal 30 S of 12000 rpm again, obtain genomic dna and RNA;
(7) forward DNA and RNA purification column to another centrifuge tube, add the DEPC aqueous solution of 50 μ l 1%, 12000 centrifugal 30 S, can obtain DNA and RNA of highly purified virus and tissue gene group, save backup in-20 DEG C.
embodiment 4: a kind of be used for animal tissues's genomic dna and RNA rapid extraction test kit and extracting method, substantially identical with example 1, difference is: adopt 0.5%(weight ratio) the diethylpyrocarbonate aqueous solution;
Lysate: EDTA 50 mM, 3% Triton X-100, lsothiocyanates 3 M, 0.9 M sodium-chlor;
Washing fluid 1:15 mM EDTA, the ethanol of volume ratio 70%, pH 7.0;
Washing fluid 2: containing 85 mM Tris-HCl, the ethanol of volume ratio 80%, pH 7.0.
By the genomic dna concentration results that the method for above embodiment is extracted, see the following form 1.
Note: DNA concentration unit is ng/ μ l; Absorbance ratio is the absorbance ratio (260/280) of DNA at 260 nm and 280nm places, absorbance ratio, 1.80 time, is illustrated that the DNA purity extracted is better, is judged the purity of DNA by absorbance ratio, as can be seen from the above table, the DNA purity of extraction is better.1,2,3,4,5 be respectively use this test kit from 20 mg hearts, liver, lungs, spleen, kidney, extract about 1.02 μ g, 1.07 μ g, 1.18 μ g, 1.08 μ g, 1.15 μ g genomes.6,7,8,9,10 be respectively use this test kit from 50 μ l whole bloods, blood plasma, serum, emulsion, nose liquid, extract about 1.18 μ g, 1.04 μ g, 1.07 μ g, 1.16 μ g, 1.11 μ g genomes.
By the geneome RNA concentration results that the method for above embodiment is extracted, see the following form 2.
| Sample number into spectrum | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| Extract concentrations | 21.65 | 23.11 | 22.33 | 22.45 | 21.38 | 23.79 | 22.18 | 21.69 | 22.35 | 21.67 |
| Absorbance ratio | 1.91 | 1.89 | 1.92 | 1.89 | 1.90 | 1.92 | 1.91 | 1.95 | 1.90 | 1.88 |
Note: absorbance ratio is the absorbance ratio (260/280) of RNA at 260 nm and 280nm places, absorbance ratio is between 1.8 ~ 2.0, illustrate that the RNA purity extracted is better, the purity of RNA is judged by absorbance ratio, as can be seen from Table 2, absorbance, between 1.8 ~ 2.0, illustrates that the RNA purity extracted is better.
Sample number into spectrum 1,2,3,4,5 is respectively and uses this test kit from 20 mg hearts, liver, lungs, spleen, kidney, extract about 1.08 μ g, 1.16 μ g, 1.12 μ g, 1.12 μ g, 1.07 μ g genomes.Sample number into spectrum 6,7,8,9,10 is respectively the genome using this test kit to extract about 1.19 μ g, 1.11 μ g, 1.08 μ g, 1.12 μ g, 1.08 μ g from 50 μ l whole bloods, blood plasma, serum, emulsion, nose liquid.
Be more than preferred embodiments of the present invention, all equalizations done according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.The invention is not restricted to the array mode expressed by above-described embodiment.If batch production, can by the proportional amplification of above-mentioned formula.
Claims (5)
1. the rapid extraction test kit for animal tissues's genomic dna and RNA, it is characterized in that, this test kit comprises following reagent: lysate, washing fluid 1, washing fluid 2, dehydrated alcohol and weight ratio are the diethylpyrocarbonate aqueous solution of 0.5 ~ 1.5%, and wherein the composition of lysate, washing fluid 1, washing fluid 2 is respectively:
(1) lysate: EDTA 30 ~ 50 mM, the Triton X-100 of weight ratio 1 ~ 3%, lsothiocyanates 1 ~ 3 M, 0.5 ~ 0.9 M sodium-chlor;
(2) washing fluid 1:5 ~ 15 mM EDTA, the ethanol of volume ratio 70%, pH 7.0;
(3) washing fluid 2: containing 65 ~ 85 mM Tris-HCl, the ethanol of volume ratio 80%, pH 7.0.
2. the rapid extraction test kit for animal tissues's genomic dna and RNA according to claim 1, is characterized in that, wherein: the concentration of the diethylpyrocarbonate aqueous solution is 1%;
Lysate: EDTA 40 mM, the Triton X-100 of weight ratio 2%, lsothiocyanates 2 M, 0.7 M sodium-chlor;
Washing fluid 1:10 mM EDTA, the ethanol of volume ratio 70%, pH 7.0;
Washing fluid 2: containing 75 mM Tris-HCl, the ethanol of volume ratio 80%, pH 7.0.
3. utilize DNA and the RNA rapid extraction test kit described in claim 1 or 2 to extract the genomic method of animal tissues, it is characterized in that, the method comprises the following steps:
(1) animal tissues of solid state is first carried out milled processed, liquid animal tissues is directly used in extraction;
(1) get liquid animal tissues 20 μ l ~ 100 μ l, or get solid state animal tissues 20mg ~ 100 mg after grinding, add 500 μ l lysates, 70 DEG C of water-bath 3-5 min, 12000 rpm 30 S, isolate supernatant liquor;
(2) proceed in centrifuge tube by supernatant liquor, add 500 μ l dehydrated alcohols, gradation forwards on DNA and RNA purification column, centrifugal 30 S of 12000 rpm; DNA and RNA----slightly carried, outwells the waste liquid in collection tube;
(3) purification column is rinsed with 500 μ l washing fluids 1----, centrifugal 30 S of 12000 rpm, outwell the waste liquid in collection tube;
(4) rinse purification column with 600 μ l washing fluids 2, centrifugal 30 S of 12000 rpm, outwell the waste liquid in collection tube;
(5) rinse purification column with 500 μ l washing fluids 2, centrifugal 30 S of 12000 rpm, outwell the waste liquid in collection tube;
(6) 12000 centrifugal 30 S----again;
(7) forward DNA and RNA purification column to another centrifuge tube, add the 50 μ l diethylpyrocarbonate aqueous solution, centrifugal 30 S of 12000 rpm, DNA and RNA of viral and tissue gene group, save backup in-20 DEG C.
4. described DNA and RNA rapid extraction test kit according to claim 3 extracts the genomic method of animal tissues, and it is characterized in that, the animal tissues of described solid state is heart, liver, lungs, spleen or kidney; Described liquid animal tissues is whole blood, blood plasma, serum or animal secretions.
5. test kit according to claim 1 is for the application in animal tissues's genomic dna and RNA rapid extraction.
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Cited By (4)
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| CN105385680A (en) * | 2015-12-24 | 2016-03-09 | 天津脉络生物科技有限公司 | Reagent for simultaneous extraction of DNA and RNA, extraction method and application |
| CN106434633A (en) * | 2016-10-25 | 2017-02-22 | 山东艾莱普生物科技有限公司 | Quick osseous tissue RNA extraction kit |
| CN107904233A (en) * | 2017-12-31 | 2018-04-13 | 贵州大学 | A kind of method for total RNA from animal tissues extraction |
| CN111235143A (en) * | 2020-02-27 | 2020-06-05 | 上海探普生物科技有限公司 | Extraction method of virus nucleic acid |
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| CN101103109A (en) * | 2005-02-04 | 2008-01-09 | 富士胶片株式会社 | Method for separating and purifying nucleic acid |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105385680A (en) * | 2015-12-24 | 2016-03-09 | 天津脉络生物科技有限公司 | Reagent for simultaneous extraction of DNA and RNA, extraction method and application |
| CN106434633A (en) * | 2016-10-25 | 2017-02-22 | 山东艾莱普生物科技有限公司 | Quick osseous tissue RNA extraction kit |
| CN107904233A (en) * | 2017-12-31 | 2018-04-13 | 贵州大学 | A kind of method for total RNA from animal tissues extraction |
| CN111235143A (en) * | 2020-02-27 | 2020-06-05 | 上海探普生物科技有限公司 | Extraction method of virus nucleic acid |
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Application publication date: 20150826 |