CN102978105A - mRNA (messenger ribonucleic acid) quick-extraction kit - Google Patents
mRNA (messenger ribonucleic acid) quick-extraction kit Download PDFInfo
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- CN102978105A CN102978105A CN2012105687548A CN201210568754A CN102978105A CN 102978105 A CN102978105 A CN 102978105A CN 2012105687548 A CN2012105687548 A CN 2012105687548A CN 201210568754 A CN201210568754 A CN 201210568754A CN 102978105 A CN102978105 A CN 102978105A
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Abstract
The invention relates to an mRNA (messenger ribonucleic acid) quick-extraction kit and a preparation method thereof. The kit comprises a separation device for solid-liquid separation, wherein the separation device at least comprises a layer of cellulose solid-phase carrier. The kit also comprises at least one tube containing a cell or tissue lysis solution, at least one tube containing a biotinylated Oligo (dT) probe, at least one tube containing a combination buffer solution, and at least one tube containing an elution buffer solution. The kit is simple, quick and efficient to operate, and is suitable for separating and extracting mRNA in a biological sample. The invention is mainly characterized in that the separation device uses cellulose as the solid-phase carrier, a fusion protein CBD-SA is used as a biological bridging agent, and a biologically-specific coupling method is used instead of the traditional physical adsorption method and chemical bonding method to activate the cellulose solid-phase carrier, so that the activated solid-phase carrier can be efficiently combined with the biotinylated reagent to be used for separating protein or nucleic acid.
Description
Technical field
The present invention relates to a kind of mRNA rapid extraction test kit and preparation method thereof, belong to biological technical field.
Background technology
MRNA is the abbreviation of messenger RNA, or is called messenger RNA(mRNA).MRNA via transcribing, with corresponding hereditary message, provides required message for next step is translated into protein by DNA.At present, along with molecular biological growing, the complete mRNA of high efficiency extraction is cDNA library structure, EST order-checking, Northern dot hybridization, microarray gene expression analysis and the committed step of implementing functional genome's application such as quantitative PCR from eukaryotic cell or tissue.In Mammals, a large amount of RNA molecules is that the form with tRNA and ribosome-RNA(rRNA) exists, and mRNA only accounts for the 1-5% of total RNA.
Traditional mRNA extracts and comprised for two steps; At first, the methods such as employing Trizol are extracted total RNA from cell or tissue, and then utilize Oligo (dT) cellulose column purified mRNA.Because the mRNA end contain many poly (A)+, when total RNA flow path Oligo (dT) Mierocrystalline cellulose, under the high-salt buffer effect, mRNA is by special being adsorbed on Oligo (dT) cellulose column, in low salt concn or distilled water, mRNA can be washed, and through twice Oligo (dT) cellulose column, can obtain purer mRNA.The shortcoming of this method is length consuming time, introduces the organic reagents such as phenol, chloroform, and owing to RNA is degraded easily, so yield is lower.
Along with the development of magnetic resolution carrier, in mRNA extracts, produced magnetic catch-hybridization technique.In addition, utilize the compatible reaction platform of Streptavidin-vitamin H, in succession occurred adopting single stage method to extract the report of the mRNA such as animal tissues, plant tissue, eukaryotic cell, prokaryotic cell prokaryocyte.Magnetic catch-hybridization technique is compared with traditional mRNA extracting method, has quick, simple to operate and efficient advantage
[This mainly be because, avoided the loss of the target molecule that the step such as phenol-chloroform extracting and precipitation causes in the traditional method, effectively suppressed simultaneously the Degradation of the inside and outside RNase of cell, therefore can improve the output of mRNA.At present, the commercially available magnetic ball that is used for the mRNA extraction mainly contains two classes, and a class is Oligo (dT) 25 magnetic microspheres, and a class is streptavidin paramagnetic particle (Streptavidin Paramagnetic Particles, but price is all relatively expensive SA-PMPS).And up to the present, all methods have all adopted chemical process that Oligo (dT) or SA are fixed on the solid phase carrier bar none.Therefore this research is devoted to study a kind of simple to operate, safe and reliable, efficient stable, need not be introduced the mRNA extracting method of chemical reagent.
Biological immobilization technology is by the solid-phase matrix material, the target molecule in the liquid phase is caught in surface adsorption or combination, by gravity, pressure, centrifugal, filtration etc., can easily unconjugated liquid phase be separated with solid phase through a step or multistep operation, thereby obtain pure molecules of interest.
Cellulose binding domain (CBD) is the integral part of cellulase and hemicellulase gene, has high-affinity with Mierocrystalline cellulose, as bridging agent SA is fixed in Mierocrystalline cellulose filter paper surface with this, because the existence of CBD, can effectively reduce sterically hinderedly, and this coupling is the biologic specificity coupling, difficult drop-off, organic reagent can be do not introduced yet, the biological activity of SA can better be guaranteed.Simultaneously, adopt commercially available Mierocrystalline cellulose filter paper, need not complicated processing, can become the solid phase carrier with specific function with the activation of CBD-SA biologic specificity, cost is little, and is easy to operate, is beneficial to commercialization scale operation.
Summary of the invention
The objective of the invention is provides a kind of easy, efficient, safe novel method for the extraction of mRNA, relate to fusion rotein CBD-S to the activation of the biologic specificity of Mierocrystalline cellulose solid phase carrier, adopt the solid phase carrier of this bioactivation to be used for method and the test kit that mRNA extracts.
Principal character of the present invention is: utilize fusion rotein CBD-SA as biological bridging agent, replace traditional physisorphtion and chemical bonding with the biologic specificity coupling method, make Mierocrystalline cellulose solid phase carrier activation, the solid phase carrier after the activation can high-level efficiency in conjunction with biotinylated reagent.Therefore, take biotinylated Oligo (dT) as probe, with in the lysate of the biological sample such as cell or tissue with the mRNA hybridization of Poly (A) tail, the high-affinity of recycling streptavidin-vitamin H, can successfully angle from tissue or cell pyrolysis liquid and get mRNA, schematic diagram is seen Fig. 1.
MRNA rapid extraction test kit provided by the invention comprises that a cover is used for the tripping device that solid, liquid is separated, and comprises one deck Mierocrystalline cellulose solid phase carrier at least in the described tripping device; This test kit also comprises following all compositions:
1) have at least a pipe to be the cell or tissue lysate;
2) have at least a pipe to be biotinylation Oligo (dT) probe;
3) have at least a pipe to be binding buffer liquid;
4) have at least a pipe to be elution buffer.
Described Mierocrystalline cellulose solid phase carrier is that the surface is by the solid-phase matrix of fusion rotein CBD-SA bioactivation, specifically utilize fusion rotein CBD-SA as biological bridging agent, replace traditional physisorphtion and chemical bonding with the biologic specificity coupling method, make the activation of Mierocrystalline cellulose solid phase carrier, can be high efficiency in conjunction with biotinylated reagent after the activation.
Described Mierocrystalline cellulose solid phase carrier is Mierocrystalline cellulose filter paper, absorbent cotton and other acceptable natural or modify after the Mierocrystalline cellulose solid phase material.
The tripping device that described solid, liquid is separated is any one in the following articles for use:
1) double-deck micro centrifugal pipe is comprised of be separated pipe and outer field centrifuge tube of the solid, liquid of internal layer, is microporous membrane or mesh like structure in the lower end of separator tube, and the Mierocrystalline cellulose filter paper layer is fixed in the separator tube, and the liquid collecting that will flow through is in centrifuge tube;
2) little separator column is comprised of cylinder, liquid outlet and mesh like structure, and screen cloth allows liquid to flow through and Mierocrystalline cellulose filter paper is fixed in the cylinder, and liquid is separated with solid phase;
3) a kind of microfilter is comprised of millipore filtration, mocromembrane support plate and filter.
Advantage of the present invention and beneficial effect:
Method provided by the invention can make things convenient for, separation and Extraction mRNA from the biological samples such as tissue or cell promptly, and need not introduce any chemical reagent; And, the Mierocrystalline cellulose solid phase carrier of the CBD-SA bioactivation that the present invention relates to, can adopt commercially available Mierocrystalline cellulose filter paper, absorbent cotton and other acceptable natural or modify after the Mierocrystalline cellulose solid phase material be starting material, the CBD-SA that adopts the biotechnology bacterial strain to express carries out biological sensitization, and easy and simple to handle, cost is low, and avoided introducing chemical reagent in the traditional chemical method safer, environmental protection.
Description of drawings
Fig. 1 is the schematic diagram that mRNA extracts.
Fig. 2 is surface structure and the principle of work synoptic diagram of several frequently seen solid, liquid phase-separating device
A among the figure, a kind of little separator column, formed by cylinder 1, liquid outlet 2 and mesh like structure 3, add the cell or tissue lysate supernatant with Oligo (dT) combination, liquid phase is separated fully with solid phase, the mRNA with Oligo (dT) combination in the lysate supernatant is adsorbed on the solid phase, and waste liquid then is collected in the test tube;
B among the figure, a kind of double-deck micro centrifugal pipe, formed by be separated pipe 4 and outer field centrifuge tube 5 of the solid, liquid of internal layer, microporous membrane or mesh like structure 6 in the lower end of separator tube, can allow liquid to pass through, the cell or tissue lysate supernatant of adding and Oligo (dT) combination, the mRNA with Oligo (dT) combination in the lysate supernatant is combined with solid phase, be trapped in the separator tube, the liquid that passes through is collected in the centrifuge tube;
C among the figure, a kind of microfilter is comprised of millipore filtration 7, mocromembrane support plate 8 and filter 9; With the cell or tissue lysate supernatant of Oligo (dT) combination after the Mierocrystalline cellulose solid phase carrier of bioactivation is combined, the mRNA with Oligo (dT) combination in the lysate supernatant is combined with solid phase, be trapped in the strainer with solid phase, waste liquid then by strainer, is collected in the test tube; Open filter and take out solid phase, with the ddH of RNase-free
2O wash-out solid phase can obtain mRNA.
Fig. 3 is that the RT-PCR of goal gene analyzes.
M: standard molecular weight DNA DL2000 is respectively 2000bp, 1000bp, 750bp, 500bp, 250bp, and 100bp from top to bottom; 1: the house-keeping gene that from the rat heart tissue, increases behind the separating mRNA-GAPDH gene; 2: the rat Langerhans islet plain gene that from RIN-m5F clone, increases behind the separating mRNA.
Embodiment
The composition of mRNA rapid extraction test kit provided by the invention comprises that a cover is used for the tripping device that solid, liquid is separated; At least comprise one deck Mierocrystalline cellulose solid phase carrier in the described tripping device; This test kit also comprises following all compositions:
1) have at least a pipe to be the cell or tissue lysate;
2) have at least a pipe to be biotinylation Oligo (dT) probe;
3) have at least one the pipe for binding buffer liquid (75mMNaCl, the 8.5mM Trisodium Citrate, pH7.2);
4) have at least a pipe to be elution buffer (Nuclease-Free Water).
The described tripping device that is separated for solid, liquid can be as shown in Figure 2 any:
A among the figure, a kind of little separator column, formed by cylinder 1, liquid outlet 2 and mesh like structure 3, add the cell or tissue lysate supernatant with Oligo (dT) combination, liquid phase is separated fully with solid phase, the mRNA with Oligo (dT) combination in the lysate supernatant is adsorbed on the solid phase, and waste liquid then is collected in the test tube;
B among the figure, a kind of double-deck micro centrifugal pipe, formed by be separated pipe 4 and outer field centrifuge tube 5 of the solid, liquid of internal layer, microporous membrane or mesh like structure 6 in the lower end of separator tube, can allow liquid to pass through, the cell or tissue lysate supernatant of adding and Oligo (dT) combination, the mRNA with Oligo (dT) combination in the lysate supernatant is combined with solid phase, be trapped in the separator tube, the liquid that passes through is collected in the centrifuge tube;
C among the figure, a kind of microfilter is comprised of millipore filtration 7, mocromembrane support plate 8 and filter 9; With the cell or tissue lysate supernatant of Oligo (dT) combination after the Mierocrystalline cellulose solid phase carrier of bioactivation is combined, the mRNA with Oligo (dT) combination in the lysate supernatant is combined with solid phase, be trapped in the strainer with solid phase, waste liquid then by strainer, is collected in the test tube; Open filter and take out solid phase, with the ddH of RNase-free
2O wash-out solid phase can obtain mRNA.
The concrete preparation process of described test kit and each reagent thereof such as following each example.
The activation of embodiment 1, Mierocrystalline cellulose filter paper and assembling (being the Mierocrystalline cellulose solid phase carrier preparation of bioactivation)
At first prepare fusion rotein CBD-SA (referring to Chinese patent CN101640085B).
Then, under 2-8 ℃, get a certain amount of Mierocrystalline cellulose filter paper bar, add the fermentation thalline lysate of recombination fusion protein CBD-SA, vibration fully after the reaction with the repeatedly drip washing 8 times of PBS damping fluid, then room temperature is dried, and gets final product.Take the double-deck micro centrifugal pipe of Fig. 2 B as example, the Mierocrystalline cellulose filter paper of bioactivation is cut into circle with the inner tube appropriate diameter, tiling places the bottom of internal layer separator tube, the upper end of microporous membrane or mesh like structure, thickness is 1mm, places approximately 5 metafiltration paper.
Activation and the assembling of embodiment 2 Mierocrystalline cellulose absorbent cotton
With reference to embodiment 1, under 2-8 ℃, get a certain amount of absorbent cotton, add the fermentation thalline lysate of recombination fusion protein CBD-SA, vibration fully after the reaction with the repeatedly drip washing 8 times of PBS damping fluid, then room temperature is dried, and is pressed into sheet and gets final product.The absorbent cotton of above-mentioned bioactivation is cut into circle with the inner tube appropriate diameter, and tiling is the fixing bottom of internal layer separator tube closely, and the upper end of microporous membrane or mesh like structure is about thick 2mm, gets final product.
The extraction of mRNA in embodiment 3 tissue samples
To dissect behind the rat anesthesia, get 50mg heart tissue and chopping, place lysate (4M guanidinium isothiocyanate, 25mM Trisodium Citrate and 1% beta-mercaptoethanol, pH7.0) homogenate is pulverized in, then add 1mL binding buffer liquid (75mM NaCl, the 8.5mM Trisodium Citrate, pH7.2), put upside down mixing, then add 60pM biotinylation Oligo (dT) probe, fully mixing is hatched 5min in 70 ℃.Subsequently in room temperature, 12000rpm, centrifugal 10min carefully collects supernatant, and being transferred to above-mentioned activation has in the internal layer separator tube of embodiment 1 of Mierocrystalline cellulose filter paper, the centrifugal 1min of room temperature 12000rpm.Waste liquid in the reject outer tube; Add SSC damping fluid 600 μ L in the inner tube, the centrifugal 30-60s of room temperature 12000rpm, the waste liquid in the reject collection tube repeats this step 2 time.At last inner tube is placed the outer tube of new Nuclease-Free, add 50 μ LNuclease-Free Water in the filter paper bar center of inner tube bottom, room temperature is placed 2min, the then centrifugal 1min of 12000rpm, collect the mRNA of wash-out, carry out immediately RT-PCR experiment or-70 ℃ of preservations.
The mRNA that separates is carried out RT-PCR amplification GAPDH gene, amplimer be (GAPDH1:5 '-TGA GTG GAC CTT TCT TTG AAG-3 ', GAPDH2:5 '-CAT GTA GGC CAT GAG GTC CAC-3 '), program is: 94 ℃ of 5min; 94 ℃ of 1min, 54 ℃ of 45s, 72 ℃ of 1min, 25 circulations; 72 ℃ of 10min; 4 ℃+∞.Subsequently amplified production is carried out gel electrophoresis analysis, 10% agarose gel electrophoresis can amplify goal gene shown in Fig. 3 swimming lane 1.
The extraction of mRNA in embodiment 4 cell samples
Get the 2-5 that centrifugal collection cultivates * 10
6Individual RIN-m5F cell, clean cell 1 time with 1 * PBS of pre-cold sterilization after, add 400 μ L lysates, transfer in the 1.5mL centrifuge tube after making cell suspension, with 1mL syringe pressure-vaccum 5 times repeatedly, make the complete cracking of cell.Add 800 μ L binding buffer liquid, put upside down and add 50pM biotinylation Oligo (dT) probe behind the mixing, abundant mixing is in 70 ℃ of incubation 5min.The centrifugal 10min of room temperature 12000rpm carefully collects supernatant subsequently, and being added to above-mentioned activation has in the internal layer separator tube of embodiment 2 of absorbent cotton, the centrifugal 1min of room temperature 12000rpm, the waste liquid in the reject outer tube; Add SSC damping fluid 600 μ L in the inner tube, the centrifugal 30-60s of room temperature 12000rpm, the waste liquid in the reject collection tube repeats this step 2 time.At last inner tube is placed the outer tube of new Nuclease-Free, add 50 μ LNuclease-Free Water in the filter paper bar center of inner tube bottom, room temperature is placed 2min, the then centrifugal 1min of 12000rpm, collect the mRNA of wash-out, carry out immediately RT-PCR experiment or-70 ℃ of preservations.
With the mRNA that separates carry out RT-PCR amplification insulin gene (1386bp) amplimer for (insulin1:5 '-CAA TCA TAG ACC ATC AGC AAG C-3 ', insulin2:5 '-AAG ATA GGCAGG GTT GAG GC-3 '), program is: 943min; 94 ℃ of 45s, 60 ℃ of 45s, 72 ℃ of 1min, 30 circulations; 72 ℃ of 10min; 4 ℃+∞.Subsequently amplified production is carried out gel electrophoresis analysis, 10% agarose gel electrophoresis amplifies goal gene shown in Fig. 3 swimming lane 2.
Claims (4)
1. a mRNA rapid extraction test kit is characterized in that this test kit comprises that a cover is used for the tripping device that solid, liquid is separated, and comprises one deck Mierocrystalline cellulose solid phase carrier at least in the described tripping device; This test kit also comprises following all compositions:
1) have at least a pipe to be the cell or tissue lysate;
2) have at least a pipe to be biotinylation Oligo (dT) probe;
3) have at least a pipe to be binding buffer liquid;
4) have at least a pipe to be elution buffer.
2. test kit according to claim 1, it is characterized in that the Mierocrystalline cellulose solid phase carrier in the described tripping device is that the surface is by the solid-phase matrix of fusion rotein CBD-SA bioactivation, specifically utilize fusion rotein CBD-SA as biological bridging agent, replace traditional physisorphtion and chemical bonding with the biologic specificity coupling method, make the activation of Mierocrystalline cellulose solid phase carrier, can be high efficiency in conjunction with biotinylated reagent after the activation.
3. test kit according to claim 1 is characterized in that described Mierocrystalline cellulose solid phase carrier is Mierocrystalline cellulose filter paper, absorbent cotton and other acceptable natural or modify after the Mierocrystalline cellulose solid phase material.
4. test kit according to claim 1 is characterized in that tripping device that described solid, liquid is separated is any one in the following articles for use:
1) double-deck micro centrifugal pipe is comprised of be separated pipe and outer field centrifuge tube of the solid, liquid of internal layer, is microporous membrane or mesh like structure in the lower end of separator tube, and the Mierocrystalline cellulose filter paper layer is fixed in the separator tube, and the liquid collecting that will flow through is in centrifuge tube;
2) little separator column is comprised of cylinder, liquid outlet and mesh like structure, and screen cloth allows liquid to flow through and Mierocrystalline cellulose filter paper is fixed in the cylinder, and liquid is separated with solid phase;
3) a kind of microfilter is comprised of millipore filtration, mocromembrane support plate and filter.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105802958A (en) * | 2016-06-01 | 2016-07-27 | 东北林业大学 | Direct plant mRNA (messenger ribonucleic acid) extraction method |
| CN105802959A (en) * | 2016-06-01 | 2016-07-27 | 东北林业大学 | Plant mRNA (messenger ribonucleic acid) extraction method |
| CN108866044A (en) * | 2018-07-23 | 2018-11-23 | 华中科技大学鄂州工业技术研究院 | A kind of method of rapidly extracting genomic DNA |
| CN109055354A (en) * | 2018-07-23 | 2018-12-21 | 华中科技大学鄂州工业技术研究院 | A kind of genomic DNA rapidly extracting kit and preparation method thereof |
| WO2019023961A1 (en) * | 2017-08-02 | 2019-02-07 | Suzhou Bofu Biomedical Limited | Method for capturing target cells or molecules in solution |
| WO2019023960A1 (en) * | 2017-08-02 | 2019-02-07 | Suzhou Bofu Biomedical Limited | Functionalized mesh and fluidic apparatus for capturing cells or molecules in solution |
| CN114672481A (en) * | 2022-04-21 | 2022-06-28 | 叶晓君 | Novel high-efficiency nucleic acid extraction method of plant fiber adsorption matrix |
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| CN101640085A (en) * | 2009-07-03 | 2010-02-03 | 南开大学 | Preparation of fibrin magnetic micrometer material for separating mRNA and application thereof |
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| CN1712547A (en) * | 2005-02-01 | 2005-12-28 | 合肥中科大生物技术有限公司 | Fluorescent quantitative RT-PCR detecting kit of 2-f(o)etoprotein (AFP)mRNA |
| CN101640085A (en) * | 2009-07-03 | 2010-02-03 | 南开大学 | Preparation of fibrin magnetic micrometer material for separating mRNA and application thereof |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105802958A (en) * | 2016-06-01 | 2016-07-27 | 东北林业大学 | Direct plant mRNA (messenger ribonucleic acid) extraction method |
| CN105802959A (en) * | 2016-06-01 | 2016-07-27 | 东北林业大学 | Plant mRNA (messenger ribonucleic acid) extraction method |
| WO2019023961A1 (en) * | 2017-08-02 | 2019-02-07 | Suzhou Bofu Biomedical Limited | Method for capturing target cells or molecules in solution |
| WO2019023960A1 (en) * | 2017-08-02 | 2019-02-07 | Suzhou Bofu Biomedical Limited | Functionalized mesh and fluidic apparatus for capturing cells or molecules in solution |
| US11525829B2 (en) | 2017-08-02 | 2022-12-13 | Hemosmart Medical Technology Ltd. | Method for capturing target cells or molecules in solution |
| CN108866044A (en) * | 2018-07-23 | 2018-11-23 | 华中科技大学鄂州工业技术研究院 | A kind of method of rapidly extracting genomic DNA |
| CN109055354A (en) * | 2018-07-23 | 2018-12-21 | 华中科技大学鄂州工业技术研究院 | A kind of genomic DNA rapidly extracting kit and preparation method thereof |
| CN114672481A (en) * | 2022-04-21 | 2022-06-28 | 叶晓君 | Novel high-efficiency nucleic acid extraction method of plant fiber adsorption matrix |
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Application publication date: 20130320 |