CN118109459B - Kit for extracting RNA (ribonucleic acid) by magnetic bead method without proteinase k treatment and extraction method - Google Patents
Kit for extracting RNA (ribonucleic acid) by magnetic bead method without proteinase k treatment and extraction method Download PDFInfo
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Abstract
The invention discloses a kit for extracting RNA by a magnetic bead method without proteinase k treatment and an extraction method, wherein the kit comprises a lysate, a binding solution, a washing solution I, DNase I digestion solution, a recombination solution, a washing solution II and an eluent.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a kit for extracting RNA by a magnetic bead method without proteinase k treatment and an extraction method.
Background
The RNA extraction technology mainly comprises an organic solvent extraction technology (Trizol method), a silica gel membrane adsorption technology (silica gel membrane centrifugal column extraction method) and a magnetic bead adsorption technology (magnetic bead extraction method). The Trizol method uses toxic reagents such as benzene, phenols, chloroform and the like, and has complicated manual operation and long time consumption; the silica gel membrane centrifugal column method is simple to operate, but the extraction process needs repeated centrifugation, so that the high-flux and automatic operation is inconvenient, and the processing requirement of a large number of samples is difficult to meet; the magnetic bead method RNA extraction uses superparamagnetism silicon oxide nanometer magnetic beads as carriers, and the superparamagnetism silicon oxide nanometer magnetic beads are specifically identified and combined with RNA molecules with high efficiency through functional groups modified on the surfaces of the magnetic beads. The magnetic bead method can realize the automation of the extraction process and the high-flux operation, and greatly improves the efficiency of RNA extraction.
The magnetic bead method nucleic acid extraction process generally comprises the steps of cracking, combining, washing, eluting and the like, wherein under the action of guanidine isothiocyanate, guanidine hydrochloride and an externally applied magnetic field, nucleic acid in a sample is released and combined with magnetic beads, and then nonspecifically adsorbed impurities are washed away to obtain purified nucleic acid. Animal tissue samples are protein-rich, and in order to extract high quality RNA, tissue cells are first lysed rapidly and efficiently. The existing magnetic bead method for RNA extraction is used for treating tissues by proteinase K, incubating at 55-70 ℃ for 10-20 min, and has long cleavage time and additionally adding a heating step. In addition, the magnetic bead method RNA extraction requires DNase I (deoxyribonuclease I) treatment to remove genome pollution, and the processes of recombination, washing and the like after DNase I treatment influence the RNA yield and purity, so that the RNA extracted by the existing method has low purity and yield and poor integrity.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a kit for extracting RNA by a magnetic bead method without proteinase k treatment.
In order to achieve the above purpose, the invention adopts the following technical scheme: a kit for extracting RNA by a magnetic bead method without protease k treatment comprises a lysate, a binding solution, a washing solution I, DNase I digestive juice, a recombination solution, a washing solution II and an eluent, wherein,
The lysate comprises guanidine isothiocyanate with the final concentration of 2-6M, tris-HCl with the final concentration of 10-100 mM, triton X-100 with the volume percentage of 1-10%, ethylphenyl polyethylene glycol with the volume percentage of 0.5-5%, potassium acetate with the volume percentage of 0.1-1%, rubidium chloride with the final concentration of 1-10 mM, EDTA with the final concentration of 10-100 mM and TCEP with the final concentration of 10-100 mM;
The binding liquid is absolute ethyl alcohol;
the washing liquid I comprises Tris-HCl with the final concentration of 10-100 mM, EDTA with the final concentration of 5-10mM and ethanol with the volume percentage of 20-80 percent;
The DNase I digestion solution comprises DNase I and 10 xDNase I buffer solution, wherein the 10 xDNase I buffer solution comprises Tris-HCl with the final concentration of 100-500 mM, mgCl 2 with the final concentration of 10-100 mM and CaCl 2 with the final concentration of 10-100 mM;
The recombination liquid comprises 1-10% of triton X-100 by volume percent, 0.5-5% of ethylphenyl polyethylene glycol by volume percent, 10-100 mM% of Tris-HCl by final concentration, 5-10 mM% of EDTA by final concentration and 20-80% of ethanol by volume percent;
wash II was 80% ethanol.
As a specific embodiment, the DNase I concentration in the DNase I digestion solution is 1-3U/. Mu.l.
In a specific embodiment, the pH of the lysate, the washing solution I and the recombination solution is 5 to 8.
As a specific embodiment, the eluent is DEPC water.
In a specific embodiment, the DNase I digestion liquid is composed of DNase I, 10×dnase I buffer and DEPC water.
Another object of the present invention is to provide a method for extracting RNA using the above kit for extracting RNA using a magnetic bead method without proteinase k treatment, comprising the steps of:
Step 1, placing a tissue sample into a centrifuge tube, homogenizing, and adding a lysis solution to fully lyse the tissue sample;
Step 2, adding an equal volume of binding solution into the cracking product obtained in the step 1, uniformly mixing, adding magnetic beads, uniformly mixing again, and standing for 5min;
step3, placing the magnetic beads on a magnetic frame, and after the magnetic beads are completely adsorbed, absorbing and discarding liquid;
Step 4, adding the washing liquid I, uniformly mixing, then placing on a magnetic frame, absorbing and discarding the liquid after the magnetic beads are completely adsorbed, and airing at room temperature for 5min;
step 5, adding DNase I digestive juice to remove genome;
Step 6, adding the recombination liquid, uniformly mixing, standing for 5min, then placing on a magnetic frame, and after the magnetic beads are completely adsorbed, absorbing and discarding the liquid;
Step 7, adding the washing liquid II, uniformly mixing, then placing the mixture on a magnetic rack, and absorbing and discarding the washing liquid after the magnetic beads are completely adsorbed;
step 8, repeating the step7 once;
Step 9, uncovering the cover, airing at room temperature for 10-15 min, adding eluent into the centrifuge tube, uniformly mixing, heating at 50-60 ℃ for 5-10 min, placing the centrifuge tube on a magnetic frame, and transferring the eluent into a new centrifuge tube for standby after the magnetic beads are completely adsorbed.
As a specific embodiment, the magnetic beads are hydroxyl silicon oxide magnetic beads, the particle size is 300-400 nm, and the concentration of the magnetic bead solution is 50mg/ml.
Due to the application of the technical scheme, compared with the prior art, the invention has the following advantages: the invention provides a kit for extracting RNA by a magnetic bead method without proteinase k treatment, which ensures that the extracted RNA has high quality and good integrity by optimizing the formulas of tissue RNA lysate, DNase I digestive juice, recombination liquid and washing liquid I, and can be directly applied to downstream experiments such as RT-PCR, qPCR, second-generation sequencing and the like.
Drawings
FIG. 1 is an agarose gel electrophoresis of RNA extracted from different tissues of mice in examples 1 to 10 and comparative example according to the present invention.
Detailed Description
The technical scheme of the invention is further described below with reference to the accompanying drawings and specific embodiments.
The invention provides a kit for extracting RNA (ribonucleic acid) by a magnetic bead method without protease k treatment, which comprises a lysate, a binding solution, a washing solution I, DNase I digestion solution, a recombination solution, a washing solution II and an eluent, wherein the lysate comprises guanidine isothiocyanate with the final concentration of 2-6M, tris-HCl with the final concentration of 10-100 mM, triton X-100 with the volume percentage of 1-10%, ethylphenyl polyethylene glycol with the volume percentage of 0.5-5%, potassium acetate with the volume percentage of 0.1-1%, rubidium chloride with the final concentration of 1-10 mM, EDTA with the final concentration of 10-100 mM, TCEP with the final concentration of 10-100 mM and pH of 5-8;
The binding liquid is absolute ethyl alcohol;
The washing liquid I comprises Tris-HCl with the final concentration of 10-100 mM, EDTA with the final concentration of 5-10 mM, ethanol with the volume percentage of 20-80 percent and pH of 5-8;
The DNase I digestion solution comprises DNase I and 10 xDNase I buffer solution, wherein the concentration of DNase I is 1-3U/. Mu.l, and the 10 xDNase I buffer solution comprises Tris-HCl with the final concentration of 100-500 mM, mgCl 2 with the final concentration of 10-100 mM and CaCl 2 with the final concentration of 10-100 mM;
The recombination liquid comprises 1-10% of triton X-100 by volume percent, 0.5-5% of ethylphenyl polyethylene glycol by volume percent, 10-100 mM% of Tris-HCl by final concentration, 5-10 mM% of EDTA by final concentration, 20-80% of ethanol by volume percent and pH value of 5-8;
washing solution II is 80% ethanol;
The eluent is DEPC water, and the kit does not contain proteinase k.
Wherein DNase I is DNase I; triton X-100 is also known as octoxynol; EDTA is ethylenediamine tetraacetic acid; TCEP is tris (2-chloroethyl) phosphate; the DEPC water is water treated by diethyl pyrocarbonate.
The method for extracting RNA by using the kit for extracting RNA without proteinase k treatment by using the magnetic bead method comprises the following steps of:
Step 1, placing a tissue sample into a centrifuge tube, homogenizing, and adding a lysis solution to fully lyse the tissue sample;
Step 2, adding an equal volume of binding solution into the cracking product obtained in the step 1, uniformly mixing, adding magnetic beads, uniformly mixing again, and standing for 5min;
step3, placing the magnetic beads on a magnetic frame, and after the magnetic beads are completely adsorbed, absorbing and discarding liquid;
Step 4, adding the washing liquid I, uniformly mixing, then placing on a magnetic frame, absorbing and discarding the liquid after the magnetic beads are completely adsorbed, and airing at room temperature for 5min;
step 5, adding DNase I digestive juice to remove genome;
Step 6, adding the recombination liquid, uniformly mixing, standing for 5min, then placing on a magnetic frame, and after the magnetic beads are completely adsorbed, absorbing and discarding the liquid;
Step 7, adding the washing liquid II, uniformly mixing, then placing the mixture on a magnetic rack, and absorbing and discarding the washing liquid after the magnetic beads are completely adsorbed;
step 8, repeating the step7 once;
And 9, uncovering, airing at room temperature for 10-15 min ℃, adding eluent into the centrifuge tube, uniformly mixing, heating at 55 ℃ for 5min, placing the centrifuge tube on a magnetic frame, and transferring the eluent into a new centrifuge tube for standby after the magnetic beads are completely adsorbed.
As a specific embodiment, the magnetic beads are hydroxyl silicon oxide magnetic beads, the particle size is 300-400 nm, and the concentration of the magnetic bead solution is 50mg/ml.
The invention is described in detail below by means of several examples:
Example 1
This example is for the extraction of RNA from mouse heart tissue.
The kit used in this example consisted of lysate, conjugate solution, wash solution I, DNase I digest, re-conjugate solution, wash solution II and eluent.
Wherein: the lysate consists of the following components: guanidine isothiocyanate with a final concentration of 2M, tris-HCl with a final concentration of 50mM, triton X-100 with a volume percentage of 3%, ethylphenyl polyethylene glycol with a volume percentage of 1%, potassium acetate with a volume percentage of 0.5%, rubidium chloride with a final concentration of 2 mM, EDTA with a final concentration of 50mM, TCEP with a final concentration of 50mM, and pH of 6.2;
The binding liquid is absolute ethyl alcohol;
Washing solution I consists of Tris-HCl with a final concentration of 50mM, EDTA with a final concentration of 5mM, ethanol with a volume percentage of 30%, and pH of 5;
the DNase I digestion solution consists of DNase I, 10 XDNase I buffer solution and DEPC water, wherein the concentration of DNase I is 2U/mu l, and the 10 XDNase I buffer solution consists of the following components: tris-HCl with a final concentration of 120mM, mgCl 2 with a final concentration of 20mM, caCl 2 with a final concentration of 20 mM;
The recombination liquid consists of the following components: 2% of triton X-100, 1% of ethylphenyl polyethylene glycol, 20 mM% of Tris-HCl, 5 mM% of EDTA, 30% of ethanol and pH 7.2;
Washing solution II is 80% ethanol; the eluent is DEPC water, and the kit does not contain proteinase k.
DNase I was purchased from Semer Feichi technologies Co., ltd and is assigned the product number EN0521.
The process for extracting RNA of the mouse heart tissue adopts the following steps:
Step 1, taking 20mg of mouse hearts, and immediately adding 500 μl of lysate after homogenization to fully lyse the mice;
Step 2, adding an equal volume of 500 μl of binding solution (absolute ethanol) into the cracked product, uniformly mixing, adding 20 μl of magnetic beads, uniformly mixing again, and standing for 5 min;
step3, placing the magnetic beads on a magnetic frame, and after the magnetic beads are completely adsorbed, absorbing and discarding liquid;
step 4, adding 500 μl of washing liquid I, mixing, placing on a magnetic rack, absorbing and discarding the liquid after the magnetic beads are completely adsorbed, and airing at room temperature for 5 min;
Step 5, adding 300 mu lDNase I of digestion solution for genome removal, wherein DNase I digestion solution consists of 10 mu l DNase I, 30 mu l 10 XDNase I buffer solution and 260 mu l DEPC water;
step 6, adding 500 mu l of recombined liquid, uniformly mixing, standing for 5min, then placing on a magnetic frame, and absorbing and discarding the liquid after the magnetic beads are completely adsorbed;
step 7, adding 500 μl of washing liquid II, mixing, placing on a magnetic rack, and absorbing and discarding the washing liquid after the magnetic beads are completely adsorbed;
step 8, repeating the step7 once;
step 9, uncovering, airing at room temperature for 10-15min, adding 60 mu l DEPC water into a centrifuge tube, uniformly mixing, and heating at 55 ℃ for 5 min; and (3) placing the centrifuge tube on a magnetic rack, and transferring the eluent into a new centrifuge tube for standby after the magnetic beads are completely adsorbed.
Example 2
This example is for the extraction of RNA from mouse heart tissue.
The kit comprises a lysis solution, a binding solution, a washing solution I, DNase I digestion solution, a recombination solution, a washing solution II and an eluent.
Wherein: the lysate consists of the following components: guanidine isothiocyanate with a final concentration of 4M, tris-HCl with a final concentration of 70mM, triton X-100 with a volume percentage of 5%, ethylphenyl polyethylene glycol with a volume percentage of 3%, potassium acetate with a volume percentage of 0.5%, rubidium chloride with a final concentration of 5mM, EDTA with a final concentration of 70mM, TCEP with a final concentration of 60mM, and pH of 5.8;
The binding liquid is absolute ethyl alcohol;
washing solution I consists of Tris-HCl with a final concentration of 60mM, EDTA with a final concentration of 7mM, ethanol with a volume percentage of 40%, and pH of 5.8;
The DNase I digestion solution consists of DNase I, 10 XDNase I buffer solution and DEPC water, wherein the concentration of DNase I is 2.5U/. Mu.l, and the 10 XDNase I buffer solution consists of the following components: tris-HCl with a final concentration of 200mM, mgCl 2 with a final concentration of 50mM, caCl 2 with a final concentration of 50 mM;
the recombination liquid consists of the following components: 5% of triton X-100, 2% of ethylphenyl polyethylene glycol, 40 mM% of Tris-HCl, 6 mM% of EDTA, 40% of ethanol and pH of 6.8;
Washing solution II is 80% ethanol; the eluent is DEPC water, and the kit does not contain proteinase k.
In this example, the procedure for extracting RNA from the heart tissue of a mouse was the same as that in example 1, and the heart tissue of a mouse was also extracted at 20mg.
Example 3
This example is for the extraction of RNA from mouse lung tissue.
The kit used was 20mg of mouse lung tissue used in the extraction procedure as in example 1.
Example 4
This example is for the extraction of RNA from mouse lung tissue.
The kit used was 20mg of mouse lung tissue used in the extraction procedure as in example 2.
Example 5
This example is for the extraction of RNA from mouse liver tissue.
The kit comprises a lysis solution, a binding solution, a washing solution I, DNase I digestion solution, a recombination solution, a washing solution II and an eluent.
Wherein: the lysate consists of the following components: guanidine isothiocyanate with a final concentration of 6M, tris-HCl with a final concentration of 100mM, triton X-100 with a volume percentage of 8%, ethylphenyl polyethylene glycol with a volume percentage of 4%, potassium acetate with a volume percentage of 0.1%, rubidium chloride with a final concentration of 6 mM, EDTA with a final concentration of 50mM, TCEP with a final concentration of 10mM, and pH of 7.8;
The binding liquid is absolute ethyl alcohol;
Washing solution I consists of Tris-HCl with the final concentration of 20mM, EDTA with the final concentration of 5mM, ethanol with the volume percentage of 20%, and the pH value of 5.5;
The DNase I digestion solution consists of DNase I, 10 XDNase I buffer solution and DEPC water, wherein the concentration of DNase I is 1U/mu l, and the 10 XDNase I buffer solution consists of the following components: tris-HCl with a final concentration of 300mM, mgCl 2 with a final concentration of 70mM, caCl 2 with a final concentration of 40 mM;
The recombination liquid consists of the following components: 3% of triton X-100, 1% of ethylphenyl polyethylene glycol, 60 mM% of Tris-HCl, 5 mM% of EDTA, 70% of ethanol and pH of 6.6;
Washing solution II is 80% ethanol; the eluent is DEPC water, and the kit does not contain proteinase k.
The extraction procedure was the same as in example 1, with 20mg of mouse liver tissue used in the extraction procedure.
Example 6
This example is for the extraction of RNA from mouse liver tissue.
The kit used was 20mg of mouse liver tissue used in the extraction procedure as in example 2.
Example 7
This example is for the extraction of RNA from mouse kidney tissue.
The kit used was the same as in example 5, and 20mg of mouse kidney tissue was used in the extraction.
Example 8
This example is for the extraction of RNA from mouse kidney tissue.
The kit used was the same as in example 2, and 20mg of mouse kidney tissue was used in the extraction.
Example 9
This example is for the extraction of RNA from mouse spleen tissue.
The kit used was the same as in example 5, and 20mg of mouse spleen tissue was used in the extraction.
Example 10
This example is for the extraction of RNA from mouse spleen tissue.
The kit used was the same as in example 2, and 20mg of mouse spleen tissue was used in the extraction.
Comparative example 1
This example is different from example 2 in that the kit used in this example does not contain rubidium chloride in the lysate in order to extract 20mg of RNA from heart tissue of a mouse.
The extraction procedure was the same as in example 1.
The concentration and purity of RNA of different tissues of the mice extracted in the above example were measured by the Nanodrop method, and the test results are shown in Table 1.
TABLE 1
RNA purity can be detected by OD 260/280 and OD 260/230. If the OD 260/280 ratio is between 1.9 and 2.1, the purity is considered to be better, and if the ratio is smaller than 1.8, the impurity proteins are more; the ratio is greater than 2.2, indicating that the RNA has been degraded; if the OD 260/230 ratio is between 1.8 and 2.1, the purity is considered to be better, and if the ratio is smaller than 1.8, the impurity proteins are more; a ratio greater than 2.2 indicates that the RNA has degraded.
As can be seen from Table 1, the concentration of RNA extracted by the kit of the invention is higher, the ratio of OD 260/280 is between 1.9 and 2.1, and the ratio of OD 260/230 is between 1.8 and 2.1, which indicates that the purity of RNA is also higher. From left to right in FIG. 1, agarose gel electrophoresis diagrams of RNA of different tissues of the mice extracted in examples 1 to 10 and comparative example 1 are shown in sequence, and as can be seen from FIG. 1, the RNA strips extracted by the kit of the invention are clear, the integrity is high, and no genomic DNA remains.
Furthermore, as can be seen from table 1 in combination with fig. 1, the concentration, purity and integrity of RNA extracted by the examples are higher than those of the comparative examples.
The above embodiments are provided to illustrate the technical concept and features of the present invention and are intended to enable those skilled in the art to understand the content of the present invention and implement the same, and are not intended to limit the scope of the present invention. All equivalent changes or modifications made in accordance with the spirit of the present invention should be construed to be included in the scope of the present invention.
Claims (7)
1. A kit for extracting RNA by a magnetic bead method without proteinase k treatment is characterized by comprising a lysate, a binding solution, a washing solution I, DNase I digestion solution, a recombination solution, a washing solution II and an eluent, wherein,
The lysate comprises guanidine isothiocyanate with the final concentration of 2-6M, tris-HCl with the final concentration of 10-100 mM, triton X-100 with the volume percentage of 1-10%, ethylphenyl polyethylene glycol with the volume percentage of 0.5-5%, potassium acetate with the volume percentage of 0.1-1%, rubidium chloride with the final concentration of 1-10 mM, EDTA with the final concentration of 10-100 mM and TCEP with the final concentration of 10-100 mM;
The binding liquid is absolute ethyl alcohol;
the washing liquid I comprises Tris-HCl with the final concentration of 10-100 mM, EDTA with the final concentration of 5-10mM and ethanol with the volume percentage of 20-80 percent;
The DNase I digestion solution comprises DNase I and 10 xDNase I buffer solution, wherein the 10 xDNase I buffer solution comprises Tris-HCl with the final concentration of 100-500 mM, mgCl 2 with the final concentration of 10-100 mM and CaCl 2 with the final concentration of 10-100 mM;
The recombination liquid comprises 1-10% of triton X-100 by volume percent, 0.5-5% of ethylphenyl polyethylene glycol by volume percent, 10-100 mM% of Tris-HCl by final concentration, 5-10 mM% of EDTA by final concentration and 20-80% of ethanol by volume percent;
wash II was 80% ethanol.
2. The kit for extracting RNA by a magnetic bead method without proteinase k treatment according to claim 1, wherein the concentration of DNase I in the DNase I digestion solution is 1-3U/. Mu.l.
3. The kit for extracting RNA by a magnetic bead method without proteinase k treatment according to claim 1, wherein the pH of the lysate, the washing solution I and the recombination solution is 5-8.
4. The kit for extracting RNA by a magnetic bead method without proteinase k treatment according to claim 1, wherein DEPC water is adopted as the eluent.
5. The kit for extracting RNA by a magnetic bead method without proteinase k treatment according to claim 1, wherein the DNase I digestion solution consists of DNase I, 10 XDNase I buffer and DEPC water.
6. A method for RNA extraction using the kit for extracting RNA according to claim 1, wherein the method comprises the steps of:
Step 1, placing a tissue sample into a centrifuge tube, homogenizing, and adding a lysis solution to fully lyse the tissue sample;
Step 2, adding an equal volume of binding solution into the cracking product obtained in the step 1, uniformly mixing, adding magnetic beads, uniformly mixing again, and standing for 5min;
step3, placing the magnetic beads on a magnetic frame, and after the magnetic beads are completely adsorbed, absorbing and discarding liquid;
Step 4, adding the washing liquid I, uniformly mixing, then placing on a magnetic frame, absorbing and discarding the liquid after the magnetic beads are completely adsorbed, and airing at room temperature for 5min;
step 5, adding DNase I digestive juice to remove genome;
Step 6, adding the recombination liquid, uniformly mixing, standing for 5min, then placing on a magnetic frame, and after the magnetic beads are completely adsorbed, absorbing and discarding the liquid;
Step 7, adding the washing liquid II, uniformly mixing, then placing the mixture on a magnetic rack, and absorbing and discarding the washing liquid after the magnetic beads are completely adsorbed;
step 8, repeating the step7 once;
Step 9, uncovering the cover, airing at room temperature for 10-15 min, adding eluent into the centrifuge tube, uniformly mixing, heating at 50-60 ℃ for 5-10 min, placing the centrifuge tube on a magnetic frame, and transferring the eluent into a new centrifuge tube for standby after the magnetic beads are completely adsorbed.
7. The method for extracting RNA using a kit for extracting RNA by a magnetic bead method without proteinase k treatment according to claim 6, wherein the magnetic beads are silica hydroxyls magnetic beads with a particle size of 300-400 nm and a concentration of 50mg/ml of a magnetic bead solution.
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| CN113913494A (en) * | 2021-09-14 | 2022-01-11 | 苏州锐讯生物科技有限公司 | Kit for quickly and conveniently extracting virus DNA in body fluid and extraction method |
| CN117511932A (en) * | 2023-12-20 | 2024-02-06 | 西安天隆科技有限公司 | Kit for extracting or purifying nucleic acid without proteinase K |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113913494A (en) * | 2021-09-14 | 2022-01-11 | 苏州锐讯生物科技有限公司 | Kit for quickly and conveniently extracting virus DNA in body fluid and extraction method |
| CN117511932A (en) * | 2023-12-20 | 2024-02-06 | 西安天隆科技有限公司 | Kit for extracting or purifying nucleic acid without proteinase K |
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