CN111197043A - Magnetic bead sorting solution for DNA separation and preparation method thereof - Google Patents
Magnetic bead sorting solution for DNA separation and preparation method thereof Download PDFInfo
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- 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
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- 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
- C12N15/1013—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 by using magnetic beads
Abstract
The invention provides a magnetic bead sorting solution for DNA separation, which comprises 1ml GE magnetic bead, 5-10g PEG, 12.5-50mg Tween 20, 50-125mmol NaCl, 0.25-1mmol Tris, 0.025-0.1mmol EDTA and 0.085-0.34mmol HCl based on 51ml volume of the magnetic bead sorting solution. The invention also provides a preparation method for preparing the magnetic bead sorting solution. The preparation method takes GE magnetic beads as raw materials, comprises a GE magnetic bead cleaning step and a GE magnetic bead resuspension step, and has the advantages of simple and stable operation, short time consumption and low cost. The prepared magnetic bead sorting solution has the functions of DNA purification and fragmentation screening, can be kept stable for a long time, does not need further chemical modification, improves the DNA separation and purification efficiency, can replace imported magnetic beads, and is suitable for large-scale library construction production.
Description
Technical Field
The invention relates to the field of biochemical detection, in particular to a magnetic bead sorting solution for DNA separation and a preparation method thereof.
Background
The magnetic beads are wrapped with ferroferric oxide (Fe)3O4) The magnetic substance has superparamagnetism and stronger magnetic responsiveness to an applied magnetic field. The magnetic beads dispersed in the solution can be adsorbed on the inner wall of the solution container under the action of the external magnetic field, and when the magnetic field is removed, the magnetic beads are uniformly dispersed in the solution again. Based on the property, the magnetic beads can be combined with a certain component in the solution, and then the magnetic beads are adsorbed and separated by an external magnetic field to achieve the purpose of separating the component.
In order to use magnetic beads for binding DNA fragments, specific groups, such as carboxyl, hydroxyl, etc., are generally modified on the surface of magnetic bead particles. The functional groups can be selectively combined with target nucleic acid, then magnetic beads are adsorbed and collected by an external magnetic field, and finally, the target nucleic acid is separated by eluting with nuclease-free water (enzyme-free water for short) or TE buffer solution. The general procedure for separating DNA with magnetic beads is to first lyse sample DNA to form DNA fragments, then add a magnetic bead sorting system to combine the magnetic beads with the DNA fragments, then apply a magnetic field to separate the magnetic beads combined with the DNA fragments, then add an eluent to elute the DNA fragments from the magnetic beads, and finally transfer a solution containing the DNA fragments to another container to obtain purified DNA fragments.
The magnetic bead sorting system mainly comprises magnetic beads, polyethylene glycol (PEG), salt ions (NaCl) and the like. Taking the sorting system of the carboxyl magnetic beads as an example, the surfaces of the carboxyl magnetic beads are negatively charged, DNA is water-soluble, the surfaces of the carboxyl magnetic beads are negatively charged, and salt ions are water-soluble. PEG, salt ions and DNA molecules competitively bind with water molecules, resulting in dehydration of DNA molecules, change of DNA conformation and gradual aggregation to form precipitates. Under the action of salt ions, phosphate groups of the DNA and carboxyl groups on the magnetic beads form ion bridges, so that the DNA is adsorbed on the surfaces of the magnetic beads. And finally, separating the DNA by magnetic adsorption and elution.
Regarding the magnetic beads, the DNA separation magnetic beads used in the current NGS library building process mainly comprise Beckmann XP magnetic beads and Kapa magnetic beads. The two kinds of magnetic beads are imported, so that the shelf life is long, the price is high, the packaging is not flexible, and the automatic large-scale warehouse building production is not facilitated.
Disclosure of Invention
The invention aims to provide a magnetic bead sorting solution for DNA separation and a preparation method thereof, aiming at the defects of the existing imported magnetic beads in terms of shelf life and cost. The preparation method has the advantages of simple and stable operation, short time consumption and low cost, the prepared magnetic bead separation liquid can be kept stable for a long time, further chemical modification is not needed, the DNA separation and purification efficiency is improved, and the method can be used for large-scale library construction production.
Specifically, the purpose of the invention is realized by the following technical scheme:
in a first aspect, the invention provides a magnetic bead sorting solution for DNA separation, comprising 1ml GE magnetic bead, 5-10g PEG, 12.5-50mg Tween 20, 50-125mmol NaCl, 0.25-1mmol Tris, 0.025-0.1mmol EDTA and 0.085-0.34mmol HCl based on 51ml volume of the magnetic bead sorting solution.
Further, the PEG is PEG4000, PEG6000 or PEG 8000.
In a second aspect, the present invention provides a method for preparing a magnetic bead sorting solution according to the first aspect of the present invention, where GE magnetic beads are used as a raw material, the method comprising the following steps:
(1) washing GE magnetic beads: washing the GE magnetic beads with a bead wash, wherein the bead wash comprises 0.05 vol% Tween 20 and 1mM Tris-HCl;
(2) and (3) GE magnetic bead resuspension: resuspending 1ml of the washed GE magnetic beads in 29.75ml of a pre-binding buffer, adding 20ml of a 25-50% PEG stock solution and 0.25ml of a 5-20% Tween 20 stock solution, and mixing uniformly to obtain the magnetic bead sorting solution, wherein the pre-binding buffer is prepared from 25ml of a 2-5M NaCl stock solution, 0.5ml of a 0.5-2M Tris stock solution, 0.5ml of a 0.05-0.2MEDTA stock solution, 0.17ml of a 0.5-2N HCl stock solution and 3.58ml of water.
In particular embodiments of the invention, a 25-50% PEG stock solution may, for example, comprise 25%, 30%, 35%, 40%, 45% or 50% PEG, or any number therebetween.
In particular embodiments of the invention, a 5-20% tween 20 stock solution may, for example, comprise 5%, 10%, 15% or 20% tween 20, or any value in between, tween 20.
In particular embodiments of the invention, the 2-5M NaCl stock may, for example, contain 2M, 3M, 4M or 5M NaCl, or any value in between.
In particular embodiments of the invention, the 0.5-2M Tris stock may, for example, comprise 0.5M, 1M, 1.5M or 2M Tris, or any value therebetween.
In particular embodiments of the invention, the 0.05-0.2M EDTA stock may, for example, comprise 0.05M, 0.1M, 0.15M, or 0.2M EDTA, or any value therebetween.
In particular embodiments of the invention, the 0.5-2N HCl stock solution may, for example, comprise 0.5N, 1N, 1.5N, or 2N HCl, or any value therebetween.
Further, in the step (1), the washing of the GE magnetic beads comprises the steps of enabling the GE magnetic beads to be balanced for a period of time at room temperature, shaking and uniformly mixing, magnetically separating the GE magnetic beads, and discarding the supernatant; then adding a magnetic bead cleaning solution to clean the GE magnetic beads. Usually, the shaking and blending time is 30s, but the shaking and blending time can be shortened or prolonged according to the time condition.
Further, the period of equilibration is typically 30min, but not limited to 30min, and the equilibration period can be shortened or lengthened according to actual conditions. The magnetic beads are normally stored at 4 ℃, the nucleic acid enrichment capacity of the magnetic beads is low at the low temperature, and the nucleic acid enrichment capacity can be obviously improved after the magnetic beads are balanced to the room temperature.
Further, the step of adding the magnetic bead cleaning liquid to clean the GE magnetic beads comprises first cleaning and second cleaning, wherein the first cleaning comprises adding the magnetic bead cleaning liquid, shaking and mixing, magnetically separating the GE magnetic beads, and abandoning the supernatant, and the second cleaning comprises adding the magnetic bead cleaning liquid again, shaking and mixing, magnetically separating the GE magnetic beads, and abandoning the supernatant. Usually, the shaking and blending time is 30s, but the shaking and blending time can be shortened or prolonged according to actual conditions. Generally, magnetic separation of GE magnetic beads is achieved by magnetically adsorbing the GE magnetic beads for 2min, but the magnetic adsorption time can be shortened or lengthened according to actual circumstances.
Further, in the first cleaning and the second cleaning, the volume ratio of the GE magnetic bead cleaning liquid to the GE magnetic beads is 1:1, but the volume ratio of the GE magnetic bead cleaning liquid to the GE magnetic beads may be increased or decreased according to actual conditions, and the ratio of the GE magnetic bead cleaning liquid is usually increased appropriately to achieve sufficient cleaning.
Further, in the step (2), the GE magnetic bead resuspension comprises adding 1ml of pre-binding buffer solution into the cleaned GE magnetic beads, shaking and uniformly mixing to resuspend the GE magnetic beads, and centrifuging for a short time; adding the resuspended GE magnetic beads into 28.75ml of pre-binding buffer solution, and shaking and uniformly mixing; and adding a PEG stock solution and a Tween 20 stock solution, shaking and uniformly mixing to obtain the DNA separation magnetic bead.
Further, in step (2), the PEG used is PEG4000, PEG6000 or PEG8000, preferably PEG 8000.
In a second aspect, the present invention provides a magnetic DNA isolation bead obtained by the method of the first aspect of the present invention.
According to the preparation method, the washed GE magnetic beads are in a solution system which is designed by the inventor and consists of the pre-combination buffer solution, PEG and Tween 20, so that the prepared DNA separation magnetic beads have the functions of DNA purification and fragmentation screening. The pre-combination buffer is mainly salt buffer, and PEG can compete with DNA for water molecules in the system, so that the conformation of DNA molecules is changed sharply, and the DNA molecules are compressed from a linear shape to a coiled ball shape, and then are aggregated and precipitated. The inventor designs the variation range of the salt ion concentration and the variation range of the PEG concentration of the pre-combination buffer system, and can selectively precipitate DNA with different lengths by changing the salt ion concentration and the PEG concentration.
Meanwhile, PEG (polyethylene glycol) as a molecule with a specific molecular weight (such as PEG4000, PEG6000 or PEG8000) and salt ions act on the DNA molecules together to change the conformation of the DNA molecules and change the viscosity degree of a buffer system, so that magnetic beads are in a suspended state in the system and are not easy to settle, the collision probability of the magnetic beads and the DNA molecules at a spatial position is increased, and the DNA aggregation efficiency is increased. In addition, PEG has compatibility with protein, and can remove protein impurities in the sample.
The invention has the beneficial effects that:
the invention provides a method for preparing novel DNA separation magnetic beads by taking GE magnetic beads with low price as raw materials aiming at the problems of long shelf life and high price of imported DNA separation magnetic beads, and the method has the characteristics of simple and stable operation, short time consumption and low cost. The DNA separation magnetic beads prepared by the invention can be kept stable for a long time, do not need further chemical modification, improve the purification efficiency of DNA, can be used for large-scale library construction production, and meet the requirement of NGS library construction production.
Compared with the prior art, the invention has the following advantages: DNA purification and fragment screening experiments show that the enrichment effect of the DNA separation magnetic beads on DNA is better than that of imported magnetic bead products; the DNA separation magnetic bead can be used for DNA purification and fragmentation screening of a library in an NGS experiment; the DNA separation magnetic bead has low cost, and can greatly save the cost compared with the commercially available products.
Drawings
FIG. 1 shows a comparison of Agilent 4200 peak patterns of magnetic bead sorting solutions B1, B2, B3 and B4 of the present invention and Kapa magnetic beads after two-time sorting of DNAladers in the experimental examples of the present invention.
Detailed Description
The present invention will be described in further detail below with reference to specific examples and test examples, and with reference to the accompanying drawings. It should be noted that these examples and test examples are illustrative and are not intended to limit the present invention in any way.
Example 1: preparation of magnetic beads for DNA isolation
The reagents used in this example mainly included: GE magnetic beads, polyethylene glycol (PEG), Ethylene Diamine Tetraacetic Acid (EDTA), HCl, Tris (hydroxymethyl) aminomethane (Tris), Tween 20, NaCl and non-enzyme water. These reagents are commercially available.
In order to prepare DNA separation magnetic beads, stock solutions of various reagents with different concentrations are prepared, and then a magnetic bead cleaning solution and a pre-binding buffer solution are prepared. And then cleaning and resuspending the GE magnetic beads by taking the GE magnetic beads as raw materials to prepare DNA separation magnetic beads.
1. Preparation of stock solutions of various reagents:
(1) tris stock solution: 1.21g, 2.42g, 3.63g and 4.84g of Tris were weighed and dissolved in 20ml of enzyme-free water to prepare Tris stock solutions with the concentrations of 0.5M, 1M, 1.5M and 2M.
(2) EDTA stock solution: 0.335g, 0.67g, 1.005g and 1.34g EDTA were weighed and dissolved in 20ml enzyme-free water to prepare EDTA stock solutions of 0.05M, 0.1M, 0.15M and 0.2M concentrations.
(3) HCl stock solution: 0.5ml, 1ml, 1.5ml and 2ml of concentrated HCl are absorbed and slowly added into 10ml of enzyme-free water to prepare HCl stock solutions with the concentrations of 0.5N, 1N, 1.5N and 2N.
(4) NaCl stock solution: 11.69g, 17.53g, 23.38g, 29.22g NaCl was weighed and added to 100ml of enzyme-free water to prepare 2M, 3M, 4M, 5M NaCl stock solutions.
(5) Stock solution of PEG: weighing 12.5g, 15g, 20g and 25g of PEG, dissolving in 50ml of enzyme-free water to prepare PEG stock solutions with the concentrations of 25%, 30%, 40% and 50%; PEG4000, PEG6000 or PEG8000 may be selected as required.
(6) Tween 20 stock solution: pipette 50. mu.l, 100. mu.l, 150. mu.l, 200. mu.l of Tween 20 into 800-.
2. Preparing a magnetic bead cleaning solution:
first, 20ml of 10mM Tris-HCl pH 8.0 was prepared with 0.4ml of a 0.5M Tris stock and 0.147ml of a 0.5N HCl stock. A50 ml centrifuge tube is taken, 19.9ml 10mM Tris-HCl is added, then 100 mul Tween 20 is absorbed and added, and the mixture is mixed evenly to prepare 10X magnetic bead cleaning solution. Then, 1ml of the 10X magnetic bead cleaning solution was added to 9ml of the enzyme-free water, and mixed to prepare a 1X magnetic bead cleaning solution.
3. Preparation of prebinding buffer:
and (3) taking a 50ml centrifuge tube, respectively and sequentially adding 25ml of NaCl stock solution with a selected concentration, 500 mu l of Tris stock solution with a selected concentration, 500 mu l of EDTA stock solution with a selected concentration, 170 mu l of HCl stock solution with a selected concentration and 3.58ml of enzyme-free water, and uniformly mixing to prepare a pre-combination buffer solution containing NaCl, Tris, EDTA and HCl with a selected concentration.
And 4, GE magnetic bead washing:
and taking the GE magnetic bead stock solution out of the refrigerator, balancing for 30min at room temperature, and shaking and uniformly mixing for 30 s. 1ml of GE magnetic bead stock solution is added into a 1.5ml centrifuge tube, placed on a magnetic frame for 2min, and the supernatant is discarded. And (4) putting down the rack, adding 1ml of 1X magnetic bead cleaning solution, shaking and uniformly mixing for 30s, putting up the rack for 2min, and discarding the supernatant to finish the first cleaning. Then, taking off the shelf, adding 1ml of 1X magnetic bead cleaning solution, shaking and uniformly mixing for 30s, taking on the shelf for 2min, and discarding the supernatant to finish the second cleaning.
GE magnetic bead resuspension:
adding 1ml of pre-binding buffer solution which is prepared by more than 1ml and contains NaCl, Tris, EDTA and HCl with selected concentration into 1ml of cleaned GE magnetic beads, shaking and uniformly mixing for 15s to resuspend the GE magnetic beads, and centrifuging for a short time. The resuspended GE beads were added to 28.75ml of this pre-binding buffer and vortexed for 30 s. Then adding 20ml of PEG stock solution with the selected concentration and 0.25ml of Tween 20 stock solution with the selected concentration, shaking and uniformly mixing for 30s to obtain magnetic bead sorting solution, and storing at 4 ℃.
Test example: verification of separation and screening effects of magnetic bead sorting solution
In this test example, a fragment purification and screening test was performed on the DNA ladder using the magnetic bead sorting solution prepared in example 1 and a commercially available Kapa magnetic bead to compare and verify the separation effect of the magnetic bead sorting solution of the present invention.
The TE Buffer and PEG Buffer used in this test example were obtained from Solarbio and Kapa, and DNAsder was obtained from Invitrogen (Thermo Fisher) and can detect fragments of 50bp-2,500bp, DNA fragments comprising 50bp, 100bp, 150bp, 200bp, 250bp, 300bp, 350bp, 400bp, 450bp, 500bp, 550bp, 600bp, 650bp, 700bp, 750bp, 800bp and 2500 bp.
The magnetic bead sorting solution prepared in example 1 was set up in four groups of B1, B2, B3 and B4, and Kapa magnetic beads were set up in one group, each group being three replicates. The specific formulations of B1, B2, B3 and B4 are as follows:
the specific test operation flow is as follows:
1. first sorting
(1) A series of 1.5ml centrifuge tubes were taken, 2. mu.l of 0.5. mu.g/. mu.l DNAladeder and 48. mu.l of enzyme-free water were added to each centrifuge tube, 50. mu.l of the magnetic bead sorting solution or Kapa magnetic beads prepared in example 1 was added, labeled, shaken and mixed well, and subjected to instantaneous centrifugation, and allowed to stand at room temperature for 10 min.
(2) And (3) after timing is finished, placing the centrifuge tube in a magnetic frame for 2min, removing supernatant, adding 80% ethanol solution, washing twice, drying magnetic beads in the air, adding 100 mu l of TE Buffer, shaking and uniformly mixing, and standing at room temperature for 5 min.
(3) And after timing is finished, adding 60 mu l of PEG Buffer into each centrifuge tube, shaking and uniformly mixing, and standing at room temperature for 10min to obtain a supernatant.
2. Second sorting
(4) Another series of 1.5ml centrifuge tubes was prepared in advance and 20. mu.l of the magnetic bead sort solution or Kapa magnetic beads prepared in example 1 were added and labeled, respectively. And (4) after the timing of the step (3) is finished, placing another series of 1.5ml centrifuge tubes in a magnetic frame for 2min, taking 155 mu l of the supernatant obtained in the step (3) into the corresponding centrifuge tube, shaking and uniformly mixing, centrifuging for a short time, and standing for 10min at room temperature.
(5) And (3) after timing, placing the centrifuge tube in a magnetic frame for 2min, removing supernatant, washing twice with 80% ethanol, drying magnetic beads in the air, adding 22 mu l of enzyme-free water, shaking and uniformly mixing, performing instantaneous centrifugation, and standing at room temperature for 5 min.
(6) After the timing was completed, the centrifuge tube was placed in a magnetic rack for 2min, the supernatant was aspirated and added to a 1.5ml centrifuge tube with the corresponding number, the Qubit concentration value of the DNA fragment was measured with a Qubit fluorometer (Invitrogen), and the size of the DNA fragment was detected with agilent 4200, and the results are shown in table 1 and fig. 1, respectively.
Table 1: comparison of the magnetic bead sorting solutions B1, B2, B3 and B4 of example 1 with the DNA sorting effect of Kapa magnetic beads after two-time sorting of DNA ladders
As can be seen from the concentration values of the qubits in Table 1, under the condition of using the same reagent and operation process, the concentration values of the qubits obtained by twice sorting the DNA ladder by the magnetic bead sorting solution of the invention with different concentration formulas are obviously higher than those of the Kapa magnetic beads, which indicates that the magnetic bead sorting solution of the invention has better and more stable sorting effect.
As can be seen from the Agilent 4200 peak diagram in FIG. 1, the sizes of the fragments sorted by the magnetic bead sorting solution and the Kapa magnetic beads are concentrated between 300 and 400bp, which indicates that the magnetic bead sorting solution of the present invention can completely meet the requirement of NGS library construction production.
Furthermore, from a cost perspective, the magnetic bead sorting solution of the present invention is calculated to have a cost of about 2 yuan/ml, while the Kapa magnetic bead cost is about 50 yuan/ml, which is a 96% cost savings per ml compared to the magnetic bead sorting solution of the present invention.
In conclusion, the magnetic bead sorting solution disclosed by the invention can replace Kapa magnetic beads to carry out NGS warehouse building production, can also greatly reduce the production cost, and has a very considerable application prospect.
The present invention has been described above using specific examples, which are only for the purpose of facilitating understanding of the present invention, and are not intended to limit the present invention. Numerous simple deductions, modifications or substitutions may be made by those skilled in the art in light of the teachings of the present invention. Such deductions, modifications or alternatives also fall within the scope of the claims of the present invention.
Claims (9)
1. A magnetic bead sorting solution for DNA separation is characterized by comprising 1ml of GE magnetic beads, 5-10g of PEG, 12.5-50mg of Tween 20, 50-125mmol of NaCl, 0.25-1mmol of Tris, 0.025-0.1mmol of EDTA and 0.085-0.34mmol of HCl based on 51ml of the magnetic bead sorting solution.
2. The magnetic bead sorting solution of claim 1, wherein the PEG is PEG4000, PEG6000, or PEG 8000.
3. The preparation method of the magnetic bead sorting solution according to claim 1 or 2, wherein the preparation method uses GE magnetic beads as raw materials, and comprises the following steps:
(1) washing GE magnetic beads: washing the GE magnetic beads with a bead wash, wherein the bead wash comprises 0.05 vol% Tween 20 and 1mM Tris-HCl;
(2) and (3) GE magnetic bead resuspension: resuspending 1ml of the washed GE magnetic beads in 29.75ml of a pre-binding buffer solution, then adding 20ml of a 25-50% PEG stock solution and 0.25ml of a 5-20% Tween 20 stock solution, and uniformly mixing to obtain the magnetic bead sorting solution, wherein the pre-binding buffer solution is prepared from 25ml of a 2-5M NaCl stock solution, 0.5ml of a 0.5-2M Tris stock solution, 0.5ml of a 0.05-0.2M EDTA stock solution, 0.17ml of a 0.5-2N HCl stock solution and 3.58ml of water.
4. The preparation method according to claim 3, wherein in the step (1), the washing of the GE magnetic beads comprises allowing the GE magnetic beads to be in equilibrium at room temperature for a period of time, shaking and mixing the GE magnetic beads, magnetically separating the GE magnetic beads, and discarding the supernatant; and then adding the magnetic bead cleaning solution to clean the GE magnetic beads.
5. The method of claim 4, wherein the period of equilibration is 30 min.
6. The preparation method of claim 4, wherein the adding of the magnetic bead cleaning solution to clean the GE magnetic beads comprises a first cleaning and a second cleaning, wherein the first cleaning comprises adding the magnetic bead cleaning solution, oscillating and mixing, magnetically separating the GE magnetic beads, and discarding the supernatant, and the second cleaning comprises adding the magnetic bead cleaning solution again, oscillating and mixing, magnetically separating the GE magnetic beads, and discarding the supernatant.
7. The preparation method of claim 6, wherein the volume ratio of the GE magnetic bead cleaning solution to the GE magnetic beads in the first washing and the second washing is 1: 1.
8. The preparation method according to claim 1, wherein in the step (2), the GE magnetic bead resuspension comprises adding 1ml of the pre-binding buffer to the washed GE magnetic beads, shaking and mixing the mixture uniformly to resuspend the GE magnetic beads, and centrifuging the mixture for a short time; adding the resuspended GE magnetic beads into 28.75ml of the pre-binding buffer solution, and shaking and uniformly mixing; and adding the PEG stock solution and the Tween 20 stock solution, and shaking and uniformly mixing to obtain the DNA separation magnetic beads.
9. The method according to claim 3, wherein in the step (2), the water is nuclease-free water.
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| CN114480369A (en) * | 2021-12-24 | 2022-05-13 | 广东润鹏生物技术有限公司 | Magnetic bead preservation solution, preparation method and magnetic bead product |
| CN114480369B (en) * | 2021-12-24 | 2022-12-27 | 广东润鹏生物技术有限公司 | Magnetic bead preservation solution, preparation method and magnetic bead product |
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