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Kit for extracting DNA through magnetic bead method and extraction method

A magnetic bead method and kit technology, applied in the field of molecular biology, can solve problems such as the inability to rule out the influence of polysaccharides, the inability to detect the target DNA, and the interference of the test system. It achieves simple and efficient operation methods, high DNA extraction rates, and reduced interference effect

Active Publication Date: 2018-10-19
浙江迪恩生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although most of the fungal DNA extraction by the magnetic bead method is easy to operate, its disadvantage is that the influence of polysaccharides on DNA adsorption cannot be ruled out.
[0005] In addition, although there are many classic methods for the DNA extraction of organisms, these traditional methods cannot meet the needs of some tests increasingly. Some tests require DNA with high purity and do not want to contain any other impurities, such as proteins, Peptides, RNA, polysaccharides, or other non-DNA substances, as these can interfere with the test
Sometimes, although there is DNA in the sample, the target DNA cannot be detected. This may be caused by the interference of the impurities contained in the test system. Although these impurities are small in content, they still interfere with the test results. Sometimes, Even let the detection fail

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  • Kit for extracting DNA through magnetic bead method and extraction method
  • Kit for extracting DNA through magnetic bead method and extraction method
  • Kit for extracting DNA through magnetic bead method and extraction method

Examples

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Embodiment 1

[0065] Example 1: DNA Extraction Kit for Sclerotinia-producing Fungi by Magnetic Bead Method

[0066] 1.1. Magnetic bead method Sclerotia-forming fungus DNA extraction kit includes (A7-A8 samples):

[0067] Step 1: Pick the sclerotium of the cultured fungus (yeast) into a 1.5ml centrifuge tube under sterile conditions, the mass of the sclerotia should not be less than 500mg, and freeze and grind it with liquid nitrogen. Add 250 μL solution Ⅰ, then add 50 μL RNase A, and mix well.

[0068] Step 2: Add 50 μL of proteinase K, mix well, and then bathe in constant temperature water at 60°C for 20 minutes. Centrifuge at 12000rpm for 5min, and transfer the supernatant to a new tube.

[0069] Step 3: Add 150 μL of solution II, and gently turn it up and down 5-6 times. Centrifuge at 12000rpm for 5min, and transfer the supernatant to a new tube.

[0070] Step 4: Add 350 μL solution III, and gently turn it up and down to fully lyse the bacteria solution into a transparent solution. ...

Embodiment 2

[0099] Example 2: Utilize processed magnetic beads to sample DNA extraction

[0100]According to the extraction process described in Example 1, the difference is: before the magnetic beads are used for DNA extraction, the purchased magnetic beads are processed, and the processed magnetic beads are used to extract DNA. The practicality and implementation of other methods and reagents The magnetic bead extraction method in Example 1 is the same as the steps.

[0101] The magnetic beads are processed, and the processing method is as follows:

[0102] The magnetic beads were purchased from Shanghai Carboxyphene Biomedical Technology Co., Ltd. (the same batch number as Example 1), and were specially used to extract the magnetic beads of DNA. The magnetic beads were treated with diglyphosate solution (5mol / L) and Guanidine isothiocyanate solution (8mol / L) for soaking. The treatment method is as follows: soak 2 g of magnetic beads in 10 ml of bisglyphosate solution for 5-10 hours, ...

Embodiment 3

[0109] Example 3: Determination of DNA Adsorption Capacity Using Treated Magnetic Beads

[0110] Use an ultrasonic cell disruptor to disrupt the cell wall of the bacteria to be tested (Staphylococcus aureus), and the specific method of disruption is as follows:

[0111] Bacteria were scraped and made into a suspension with distilled water (10 7 spore / mL), and then place the beaker containing the suspension in the crushing instrument, put the probe of the crushing instrument into the beaker and insert it into the spore suspension, set the crushing time to 10 min in an ice bath environment, and take out the sample after cell crushing. Magnetic bead adsorption DNA detection.

[0112] In the broken sample liquid, add magnetic beads of a certain weight (but 2-5 grams) of the present invention (the processed magnetic beads in embodiment example 2), the beaker is placed on the magnetic stand to carry out the magnetic beads adsorption of DNA, After 10 minutes, take out the magnetic ...

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Abstract

The invention discloses a kit for extracting DNA through a magnetic bead method and a rapid and simple method for extracting polysaccharide fungus DNA. The kit comprises a solution (I), a solution (II), a solution (III), a solution (IV), a washing solution, eluent, and magnetic bead suspension. The solution (II) is innovatively used to remove polysaccharides of fungi, and the interference of extracellular polysaccharides is eliminated during the DNA extraction process. During the test process, the magnetic beads of the kit are preprocessed, DNA is taken as the molecular target, the adsorbing performance of magnetic beads on RNA and proteins is weakened, and the operation has the characteristic of high specificity. The kit is used to extract DNA of polysaccharide fungi, the detection is simple and convenient, and the interference of extracellular polysaccharides on DNA extraction is reduced.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a DNA extraction kit for sclerotia-producing fungi by a magnetic bead method and a method for quickly extracting DNA of sclerotia-producing fungi with strong specificity. Background technique [0002] Sclerotia is a dormant body composed of closely intertwined hyphae, and its main function is to resist adverse environments. Common sclerotia-producing fungi include Sclerotinia and Rhizoctonia, which contain a large amount of exopolysaccharides. The fungal exopolysaccharide has the characteristics of high adsorption and high viscosity, which is one of the difficulties in the separation and extraction of high-purity DNA from sclerotia-producing fungi. Molecular biological methods are now one of the essential technologies for fungal identification, and the separation and purification of DNA is the basis for molecular biological research. At present, the traditional CTAB...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1013
Inventor 黄宵戴明雁徐华莉高颖张明洲
Owner 浙江迪恩生物科技股份有限公司
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