Binding solution, kit and method for purifying nucleic acid by magnetic bead method as well as application

The technology of a kit and magnetic bead method is applied in the field of binding solution for nucleic acid purification by magnetic beads, which can solve the problems of increasing the workload of production personnel, increasing the difficulty of the operator's work, and particularly troublesome reagent production. Better effect, longer storage time effect

Inactive Publication Date: 2020-03-27
广州高盛生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, most magnetic bead extraction kits on the market are selective for samples, and the components in the lysate are relatively simple. Most of them are configured according to traditional nucleic acid extraction methods. When encountering complex and diverse samples, different kits are often required. Nucleic acid extraction increases the difficulty of the operator's work; secondly, the binding step involves the mixing of nucleic acid adsorbent and magnetic beads. Most of the binding solutions on the market contain guanidinium salts. Although guanidinium salts can promote the precipitation of nucleic acids and the interaction with magnetic beads However, most of the magneti

Method used

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  • Binding solution, kit and method for purifying nucleic acid by magnetic bead method as well as application
  • Binding solution, kit and method for purifying nucleic acid by magnetic bead method as well as application
  • Binding solution, kit and method for purifying nucleic acid by magnetic bead method as well as application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] The assembly of embodiment 1 kit

[0093] Prepare according to the formula of each liquid below:

[0094]1. Lysis solution (pH7.0-8.0): Tris-Cl (50-150mM), EDTA (1-10mM), hydrochloric acid stock (2-4M), guanidine isothiocyanate (2-4M), SDS (0.5 -1%, m / v), TritonX-100 (1-2%, v / v), Tween-20 (1-2%, v / v), NaCl (50-100mM), choose to add according to different samples DTT (1-10mM), proteinase K (1-2mg / mL);

[0095] 2. Binding solution (pH5.0-6.0): citrate buffer (50-100mM), EDTA (1-10mM), sodium acetate (100-300mM), lithium chloride (100-200mM), isopropanol (10-30%, v / v);

[0096] 3. Rinse solution I (pH7.5-8.5): Tris-Cl (1-10mM), NaCl (50-100mM), EDTA (1-5mM), guanidine hydrochloride (1-2M);

[0097] 4. Wash solution II (pH7.5-8.5): Tris-Cl (1-10mM), EDTA (1-5mM), ethanol (75-85%, v / v);

[0098] 5. Eluent (pH7.5-8.5): Tris-Cl (1-10mM), EDTA (1-5mM), glycerol (1-5%, v / v), SDS (0.01-0.05%, m / v), sodium fluoride (0.01-0.02%, m / v);

[0099] The above-mentioned lysate, bi...

Embodiment 2

[0100] Example 2 Calculation of DNA recovery rate

[0101] Using the kit described in Example 1 to calculate the DNA recovery rate, the specific steps are as follows: take 5ng, 10ng, and 20ng standard genomic DNA respectively, repeat 3 samples for each gradient, and carry out DNA extraction through the subordinate extraction process, and extract the DNA with Qubit3 .0 for quantification, calculate the DNA recovery rate before and after extraction, the quantitative results are shown in Table 1.

[0102] (1) Take the standard DNA corresponding to the above content, dilute it into 10 μl aqueous solution, add 100-150 μl lysis solution, add 100-300 μl binding solution and 10 μl magnetic beads, and incubate in a centrifuge tube at room temperature for 10 minutes (pure DNA does not go through the lysis step) ;

[0103] (2) Place the centrifuge tube on the magnetic stand for 1 min, and remove the supernatant;

[0104] (3) Remove the centrifuge tube from the magnetic stand, add 300 μ...

Embodiment 3

[0113] Example 3 Different Carrier Blood DNA Extraction

[0114] The kit in Example 1 was used to extract DNA from different carriers, and the specific steps were as follows: Take 2 μl of whole blood, dilute it with 200 μl of ultrapure water, and drop it evenly on the FTA card, qualitative filter paper and gauze, and take 3 replicates for each carrier sample. Among them, FTA card, qualitative filter paper, and gauze are all cut into samples of 0.2cm×0.2cm size for extraction:

[0115] (1) Take the above sample in a centrifuge tube, add 200-400 μl lysate, add 2 mg / mL proteinase K, and incubate at 56°C for 20 minutes;

[0116] (2) Collect the supernatant after centrifugation at 10000rpm, add 200-600μl of binding solution and 15μl of magnetic beads, incubate at room temperature for 10min, shake and mix well during the period;

[0117] (3) Place the centrifuge tube on the magnetic stand for 1 min, and remove the supernatant;

[0118] (4) Remove the centrifuge tube from the magn...

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Abstract

The invention provides a binding solution, kit and method for purifying nucleic acid by magnetic bead method as well as application. The binding solution comprises a citrate buffer solution, EDTA, sodium acetate, lithium chloride and isopropyl alcohol but does not comprise guanidine salt. The binding solution contains no guanidine salt, can coexist with magnetic beads for a long time and can fullypromote precipitation of nucleic acid and binding with magnetic beads, the low isopropyl alcohol content contributes to increase of the nucleic acid purity, and nonspecific binding of impurities withmagnetic beads is reduced.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a binding liquid, a kit, a method and an application for magnetic bead nucleic acid purification. Background technique [0002] Magnetic bead method nucleic acid extraction reagent is a new technology product combining biochemistry and nanomaterial science. The chemically modified superparamagnetic nano magnetic beads can specifically bind to nucleic acid molecules in a specific ion reaction system. Using this principle, it can be obtained from various Isolate DNA from various animal tissues, cells, and microorganisms, and apply it in various downstream research and detection fields. [0003] The magnetic bead nucleic acid extraction process is divided into four steps: lysis-binding-washing-elution. Among them, the lysis requires the use of lysate and auxiliary additives such as proteinase K, dithiothreitol (DTT), etc., the combination needs to add magnetic beads to the specific ion r...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1013C12Q2527/125
Inventor 罗深恒康贤通章戴荣
Owner 广州高盛生物科技有限公司
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