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Double-magnetic-bead method based centrifugation-free extraction kit for plasmid DNA (deoxyribonucleic acid) and use method

A technology for extracting plasmids and magnetic beads, which is applied in the field of molecular biology, can solve problems such as unsuitable development directions, and achieve the effect of complete fragments and high purity

Active Publication Date: 2016-06-08
SUZHOU ENRICHING BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional magnetic bead method still requires centrifugation steps, which is not suitable for the development direction of high-throughput automatic extraction of bacterial plasmid DNA
[0004] As mentioned in the review, the existing technology for extracting bacterial plasmid DNA by magnetic beads cannot meet the technical problems of centrifugation-free, high-throughput, and automation. Extraction method of bacterial plasmid DNA by automatic magnetic bead method

Method used

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  • Double-magnetic-bead method based centrifugation-free extraction kit for plasmid DNA (deoxyribonucleic acid) and use method
  • Double-magnetic-bead method based centrifugation-free extraction kit for plasmid DNA (deoxyribonucleic acid) and use method
  • Double-magnetic-bead method based centrifugation-free extraction kit for plasmid DNA (deoxyribonucleic acid) and use method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: The preparation steps of the impurity-removing magnetic beads are as follows:

[0037] S1: 1-5g of coated SiO 2 Add ferroferric oxide nano-magnetic balls into the mixed system of 800mL ethanol and 200mL water, add 0.1-1g triethylamine, under the protection of nitrogen, heat up to 40-80°C, add 0.5-5gKH570, mechanically stir the reaction for 6 hours, use Wash with deionized water and ethanol three times and then dry to obtain magnetic beads modified with KH570;

[0038] S2: Add 1-5g magnetic beads modified KH570 to 1000mL DMSO solution, add 0.5-1g polyethylene, 0.2-1g divinylbenzene, 1-2g acrylic alcohol, add 50mg azobisisobutyronitrile , heat up to 60-85°C, react under nitrogen protection for 4-6 hours, add 0.5-1.5g of glycidyl methacrylate, mechanically stir and continue the reaction for 12-16 hours, wash with deionized water and ethanol three times, and then dry. Obtain modified magnetic beads.

[0039]S3: Disperse 1-5g of the above-mentioned modified mag...

Embodiment 2

[0040] Embodiment 2: A kind of extraction method of centrifugation-free, double-magnetic bead method plasmid DNA extraction kit, which includes impurity removal magnetic bead suspension, extraction magnetic bead suspension, bacterial lysate, binding buffer, cleaning solution and eluent Six components, the contents of each component are as follows:

[0041] Impurity-removing magnetic bead suspension: The impurity-removing magnetic bead suspension includes 4mg / mL impurity-removing magnetic beads and the first dispersion, wherein the components of the first dispersion are as follows: 1.5M hydrochloric acid, 3M potassium carbonate, 0.03M Tris- HCl buffer, 0.15mg / mL RNase; the default solvent used in this case is deionized water.

[0042] The impurity-removing magnetic beads are prepared by the method of Example 1;

[0043] Extract magnetic bead suspension: the extracted magnetic bead suspension includes 50mg / mL silanol magnetic beads and the second dispersion liquid, the second d...

Embodiment 3

[0056] Embodiment 3: A kind of free centrifugation, double magnetic bead method plasmid DNA extraction kit extraction method, it comprises impurity removal magnetic bead suspension, extraction magnetic bead suspension, bacterial lysate, binding buffer, cleaning solution and eluent Six components, the contents of each component are as follows:

[0057] Impurity removal magnetic bead suspension: The impurity removal magnetic bead suspension includes 5mg / mL impurity removal magnetic beads and the first dispersion liquid. The components of the first dispersion liquid are as follows: 1.5M nitric acid, 2M sodium acetate, 0.025MTris-HCl buffer, 0.1mg / mL RNase;

[0058] The impurity-removing magnetic beads are prepared by the method of Example 1;

[0059] Extract magnetic bead suspension: the extracted magnetic bead suspension includes 40mg / mL silanol magnetic beads and the second dispersion liquid, the second dispersion liquid components are as follows: 0.3M sodium chloride, pH=6; ...

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Abstract

The invention relates to a double-magnetic-bead method based centrifugation-free extraction kit for plasmid DNA (deoxyribonucleic acid). The kit accommodates an impurity-removing magnetic bead suspension liquid, an extraction magnetic bead suspension liquid, a bacterium lysis solution, a binding buffer solution, a cleaning solution and an eluent which are separately subpackaged, wherein impurity-removing magnetic beads are dispersed in the impurity-removing magnetic bead suspension liquid and surfaces of the impurity-removing magnetic beads are at least modified with styrene, divinyl benzene and iminodiacetic acid; extraction magnetic beads are dispersed in the extraction magnetic bead suspension liquid and surfaces of the extraction magnetic beads are modified with silicon hydroxyl. According to development of the impurity-removing magnetic beads, impurities such as cell walls, protein and the like are removed after bacterium lysis, the plasmid DNA in a liquid supernatant is reserved, so that the centrifugation step in a traditional process is replaced, and the operation is more efficient and faster; when the kit is in use, the buffer solution, the magnetic bead suspension liquid and the like can be subpackaged in corresponding pore plates of a nucleic acid extraction instrument, and requirements for automation and high throughput are met; the extracted plasmid DNA has high purity and complete fragments.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a kit for free-centrifugation extraction of plasmid DNA based on a double magnetic bead method and a use method thereof. Background technique [0002] DNA is the basic genetic material, which plays a decisive role in protein synthesis and the growth and variation of a series of major life. Biotechnology with DNA as the research object, including a series of technologies such as DNA extraction, cloning, amplification, and sequencing, has developed rapidly in recent years. In various studies of genetic engineering, plasmid DNA plays an important role and is one of the important factors affecting the success of downstream DNA research. Therefore, it is very important to purify bacterial plasmid DNA. [0003] Most plasmid DNA extraction methods generally include cell lysis, removal of substances bound to nucleic acid, precipitation of nucleic acid, removal of impurities ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1013C12Q2563/143C12Q2563/149
Inventor 吴巧张佳斌曲峰尚春庆
Owner SUZHOU ENRICHING BIOTECH CO LTD
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