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Simple, efficient and cheap method for purifying forest soil sample DNA

A soil sample and forest soil technology, applied in the field of total DNA purification of forest soil samples, can solve the problems of high cost, complexity and time-consuming, etc., and achieve the effect of low cost, wide application range and simple operation

Inactive Publication Date: 2010-01-20
JISHOU UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These purification methods may be suitable for the purification of some soil DNA, however, these methods are either complex, time-consuming, or costly

Method used

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  • Simple, efficient and cheap method for purifying forest soil sample DNA
  • Simple, efficient and cheap method for purifying forest soil sample DNA
  • Simple, efficient and cheap method for purifying forest soil sample DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Sample Sampling

[0024] The five soil samples used in this study were all taken from the 0-10cm surface soil of Lin’an Bamboo Forest in Hangzhou, China, including three samples with high impurity content (sample 2, 4, 5) and two samples with low impurity content (sample 1, 3), all samples were taken in triplicate.

Embodiment 2

[0025] Embodiment 2: Extraction of initial DNA crude product

[0026] Step (1): The crude DNA was extracted using a slightly modified CTAB-SDS method (Zhou et al., 1996. Applied and Environmental Microbiology, 62: 316-322): 5 g of soil sample and 13.5 mL of DNA extraction buffer (100 mM Tris- HCl, 100 mM sodium EDTA [pH 8.0], 100 mM sodium phosphate [pH 8.0], 1.5M NaCl, 1% CTAB) and 100 μL proteinase K (10 mg / mL) were mixed in a centrifuge tube, and then heated at 37 °C at 225 rpm Speed ​​horizontal oscillation for 30 minutes. After shaking, add 1.5mL of 20% SDS, and then place it in a constant temperature water bath at 65°C for 2 hours. During this period, gently invert the centrifuge tube every 15 to 20 minutes, and then centrifuge the sample at 6000 rpm for 10 minutes at room temperature. minute. Take the supernatant and transfer it to another new centrifuge tube, add an equal volume of chloroform-amylisoalcohol (24:1 (v / v)) to the supernatant to mix and extract, and cent...

Embodiment 3

[0027] Embodiment 3: further purification of crude product DNA

[0028] Step (2): Use 2 mL of extraction buffer to dissolve the crude nucleic acid precipitate, pipette several times to accelerate the dissolution, then place it in a water bath at 65°C for 10 minutes, add an equal volume of chloroform-amylisoalcohol solution (chloroform:amyl isoalcohol) isoalcohol=24:1), centrifuged at 16000 rpm for 5 minutes. Take the supernatant, add 0.6 times the volume of isopropanol, let stand at room temperature for a few minutes, then centrifuge at 16,000 rpm for 10 minutes at room temperature, discard the supernatant, and wash the precipitate with pre-cooled 70% alcohol or ethanol , and the DNA obtained after drying was dissolved in 100 μL TE buffer.

[0029] Step (3): The DNA recovered in step (2) is further purified with a silica gel column. Add 3 times the volume of QIAquick Gel Extraction Kit buffer to 100 μL of crude DNA. After a few minutes, transfer to the recovery column and ce...

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PUM

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Abstract

The invention provides a method for purifying microorganism DNA in forest soil, which comprises the following steps: (1) obtaining the rough DNA of a microorganism by a conventional method; (2) dissolving the rough DNA by extracted buffer solution; after the rough DNA is subjected to water bath and isovolumetric chloroform-isoamyl alcohol solution extraction, adding isopropanol which is 0.6 time of the volume of supernatant into the supernatant to precipitate DNA sediments; washing by 70 percent of pre-cooled alcohol and dissolving in the TE buffer solution; and (3) further purifying the initially purified DNA by a silica gel column. The method can remove most of impurities in a rough product of the extracted soil microorganism DNA and obtain the DNA with high purity and can be directly used for the conventional polymerase chain reaction (PCR) which is quite sensitive for an inhabiting matter. The invention has simple operation, low cost, less time consumption and wide application range and can be used for purifying the soil DNA in various types after other prior soil direct extraction methods.

Description

technical field [0001] The invention belongs to the technical field of environmental microorganisms, and in particular relates to a simple, high-efficiency and cheap method for purifying total DNA of forest soil samples. Background technique [0002] Microorganisms play an important role in forest soils, but only small microbial populations can be cultivated using existing techniques. Molecular biology techniques used in biology provide us with new ways to study microbial communities, however, the purity of DNA obtained from microbial community samples is crucial for subsequent molecular manipulations. Many studies have reported the extraction methods of soil microbial community DNA, such as extraction methods based on SDS, extraction methods based on glass bead beating, and extraction methods based on microwaves, but the purity of DNA obtained by these methods is not high and contains a large amount of impurities ( Such as humic acid, fulvic acid), these impurities will in...

Claims

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Application Information

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IPC IPC(8): C12N15/10C07H21/04
Inventor 何兴兵韩国民林永慧肖竹平胡文勇
Owner JISHOU UNIVERSITY
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