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Kit for extracting fungal/bacterial genomic DNA based on magnetic bead technology and method thereof

A kit and genome technology, applied in the field of molecular biology, can solve problems such as difficult to realize industrial application, damage to the health of operators, cumbersome operation steps, etc., and achieve the effects of reducing physical injuries, saving costs, and good repeatability

Pending Publication Date: 2018-04-27
HANGZHOU LC BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The purity of DNA extracted by this method is high, but the operation steps are cumbersome, the workload is heavy, and the extraction efficiency is low, so it is difficult to realize industrial application
In addition, organic solvents such as phenol, chloroform and isopropanol are likely to cause environmental pollution and damage the health of operators

Method used

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  • Kit for extracting fungal/bacterial genomic DNA based on magnetic bead technology and method thereof
  • Kit for extracting fungal/bacterial genomic DNA based on magnetic bead technology and method thereof
  • Kit for extracting fungal/bacterial genomic DNA based on magnetic bead technology and method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Preparation of reagents in the fungal / bacterial genomic DNA extraction kit.

[0028] (1) Lysis solution: a mixed solution composed of 50 mM Tris-HCl, 10 mM EDTA, 0.5 M NaCl and 1% CTAB by volume. Specific preparation method: Weigh 7.88g Tris-HCl, 2.92g EDTA and 29.22g NaCl, add 800mL deionized water and stir to dissolve, add 10mL CTAB, continue to add deionized water to make up to 1L.

[0029] (2) Magnetic bead rinsing solution: ethanol solution with a volume fraction of 70%. Specific preparation method: Measure 700mL of absolute ethanol and add 300mL of deionized water, mix well.

[0030] (3) DNA eluent: 10 mM Tris-HCl solution, pH 8.0. Specific preparation method: Weigh 1.58g Tris-HCl and add deionized water to make the volume to 1L, adjust the pH value to 8.0 with concentrated hydrochloric acid.

Embodiment 2

[0032] Genomic DNA was extracted from 4 E. coli samples using the kit in Example 1.

[0033] ① Take 200 μL of bacterial liquid sample and add 200 μL of lysate, mix well, incubate at 70°C for 10 minutes, freeze at -80°C for 15 minutes, and repeat the operation once.

[0034] ②Incubate the sample again at 70°C for 10 minutes, add 200 μL of chloroform, mix well, let stand at room temperature for 1 minute, and centrifuge at 12,000 g for 5 minutes.

[0035] ③ Take 100 μL of centrifuged aqueous phase supernatant and add 100 μL of magnetic bead solution, mix well, let stand at room temperature for 5 minutes, place on a magnetic stand for 5 minutes, and discard the supernatant.

[0036] ④ Add 200 μL magnetic bead washing solution to resuspend the magnetic bead nucleic acid mixture, wash 2-3 times, and blot the residual liquid.

[0037] ⑤ Open the lid and let stand at room temperature for 5 minutes, add 50 μL of DNA eluent, mix well by pipetting, and let stand at room temperature for ...

Embodiment 3

[0043] Genomic DNA was extracted from 4 samples of yeast and fungi using the kit in Example 1.

[0044] ① Take 200 μL of bacterial liquid sample and add 200 μL of lysate, mix well, incubate at 70°C for 10 minutes, freeze at -80°C for 15 minutes, and repeat the operation once.

[0045] ②Incubate the sample again at 70°C for 10 minutes, add 200 μL of chloroform, mix well, let stand at room temperature for 1 minute, and centrifuge at 12,000 g for 5 minutes.

[0046] ③ Take 100 μL of centrifuged aqueous phase supernatant and add 100 μL of magnetic bead solution, mix well, let stand at room temperature for 5 minutes, place on a magnetic stand for 5 minutes, and discard the supernatant.

[0047] ④ Add 200 μL magnetic bead washing solution to resuspend the magnetic bead nucleic acid mixture, wash 2-3 times, and blot the residual liquid.

[0048] ⑤ Open the lid and let stand at room temperature for 5 minutes, add 50 μL of DNA eluent, mix well by pipetting, and let stand at room tempe...

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Abstract

The invention discloses a kit for extracting fungal / bacterial genomic DNA based on a magnetic bead technology and a method thereof, and belongs to the field of molecular biology. The kit comprises five components: a lysate, chloroform, magnetic beads, a magnetic bead rinsing liquid and a DNA eluant. The genomic DNA extraction includes the main steps of cracking a sample, carrying out magnetic beadadsorption and rinsing to obtain the genomic DNA. The operation method applying the kit is simple in steps, the extraction efficiency of the genomic DNA in fungi / bacteria can be greatly improved, andthe extracted DNA product has a clear and complete electrophoresis strip after PCR amplification and has good reproducibility. In addition, reagents required by the kit are conventional reagents, thecost is low, and the kit is suitable for high-flux experiments.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a kit for extracting fungal / bacterial genomic DNA based on magnetic bead technology, which can be applied to the extraction of fungal / bacterial genomic DNA in solid or liquid samples. The invention also relates to a method for extracting fungal / bacterial genome DNA in solid or liquid samples by using the kit. Background technique [0002] In recent years, the genome research of microorganisms such as fungi and bacteria has made rapid progress. Genome research will enable humans to grasp the pathogenic mechanism and laws of pathogenic microorganisms at a higher level, so as to develop new preparations, vaccines and drugs for the diagnosis, prevention and treatment of microbial infections. How to extract high-quality DNA from fungal / bacterial samples in solid or liquid is an important prerequisite for genetic testing and other molecular biology research. [0003] The phenol / imitati...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1013
Inventor 郎秋蕾梁洪殷楠楠袁丹娄奇彬李倩
Owner HANGZHOU LC BIOTECH
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