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Soil nematode DNA extraction method

A technology of soil nematodes and extraction methods, which is applied in the field of animal DNA extraction, can solve the problems of denaturing gradient gel electrophoresis comparison, etc., and achieve the effects of avoiding the amplification of non-target biological DNA, improving the extraction rate, and low cost

Inactive Publication Date: 2009-11-18
SHENYANG INST OF APPLIED ECOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Waite et al. (2003) used the DGGE method to measure the diversity of six species of grassland soil nematodes in Europe, but did not compare the results of denaturing gradient gel electrophoresis with morphological analysis. The accuracy of this method itself needs to be further verified

Method used

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  • Soil nematode DNA extraction method
  • Soil nematode DNA extraction method

Examples

Experimental program
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Effect test

Embodiment 1

[0014] Soil nematode acquisition: Wash the sample fresh soil, pour 200g of the washed fresh soil into a water basin, add water and stir well, let it stand for 1 minute, pour it into a set of mesh sieves, the upper layer is 60 mesh, the lower layer is 400 mesh, and the Invert the edge and vibrate the sampling sieve to prevent the water from filling the lower 400-mesh sieve and overflowing from the sieve, then add water to the basin and mix well, let it stand for 1 minute, pour it into the mesh sieve, repeat this 3 times, and pour the 400-mesh The sampling sieve was removed, and the mud in the nematode suspension in the 400-mesh mesh sieve was rinsed with a nozzle, poured into another beaker, and left to stand for 24 hours. The upper layer of water in the standing beaker was poured off gently, leaving only the mixture of about 30ml of lower layer of water, nematodes and mud. Shake the mixture gently, pour it into a centrifuge tube, prepare for the first centrifugation, the speed...

Embodiment 2

[0017] The difference from Example 1 is:

[0018] The extraction of soil nematodes is to wash the samples, pass through 60-mesh and 400-mesh sieves respectively, and then add excess magnesium sulfate to the samples and centrifuge at 2000 rpm for 5 minutes to extract the samples suspended in the magnesium sulfate solution.

[0019] Kill the extracted soil nematode suspension at 65°C for 2-3 minutes with gentle heat, concentrate the mildly heat-killed soil nematode suspension at a ratio of 1:4, and then add 0.4 times its volume of cell lysis buffer to carry out Freeze-thaw treatment, and then add 20 μg of proteinase K to each microliter of the concentrated soil nematode suspension, shake at 160 rpm at 38°C for 2 hours for heat preservation and digestion; then mix the heat preservation and digestion nematode solution with an equal volume of chloroform and iso The pentanol mixture was mixed at room temperature, centrifuged at 12,000 rpm for 12 minutes, the aqueous phase was extrac...

Embodiment 3

[0021] The difference from Example 1 is:

[0022] The extraction of soil nematodes is to wash the samples, pass through 60-mesh and 400-mesh sieves respectively, and then add excess magnesium sulfate to the samples and centrifuge at 2000 rpm for 5 minutes to extract the samples suspended in the magnesium sulfate solution.

[0023] Kill the extracted soil nematode suspension at 63°C for 2-3 minutes with gentle heat, concentrate the mildly heat-killed soil nematode suspension at a ratio of 1:4, and then add 0.4 times its volume of cell lysis buffer to carry out Freeze-thaw treatment, and then add 20 μg of proteinase K to each microliter of the concentrated soil nematode suspension, shake at 150 rpm at 37°C for 1.5 hours for heat preservation and digestion; then mix the heat preservation and digestion nematode solution with an equal volume of chloroform and iso The pentanol mixture was mixed at room temperature, centrifuged at 12,000 rpm for 11 minutes, the aqueous phase was extr...

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Abstract

The invention relates to an animal DNA extraction method, in particular to a soil nematode DNA extraction method. The soil nematode DNA extraction method comprises the following steps: adopting a washing-screening-magnesium sulfate gradient centrifugation method to extract nematodes from a soil sample and killing the nematodes by a heating method to obtain a nematode suspension; then, firstly adding a cell lysis buffer solution to the nematode suspension for freeze thawing treatment and then adding protease for heat preservation and digestion; and respectively adopting chloroform and isoamyl alcohol to extract DNA, NaAc and isopropanol sediment DNA and naturally drying the DNA, the NaAc and the isopropanol sediment DNA at a room temperature so as to obtain soil nematode DNA. The invention combines physical, chemical and enzyme methods to crack nematode cells, improves the extraction rate of the nematode DNA, has lower cost and is suitable for extracting the DNA of soil nematode communities.

Description

technical field [0001] The invention relates to animal DNA extraction, in particular to a soil nematode DNA extraction method. Background technique [0002] Among the numerous soil animals, nematodes are the most abundant in number and function. Because of its special shape, food specificity, relatively simple isolation and identification, and rapid response to environmental changes, it can be used as an indicator organism for the study of soil pollution effects. One of the advantages of nematodes as indicator organisms is the ease of morphological identification, but there are still many difficulties in identification to the species level. Another obstacle that affects nematodes as indicator organisms is the number of samples. Increasing the number of samples analyzed will provide more detailed spatial information; however, increasing the repetition of samples in time and space also means increasing the workload of sample analysis. Due to the limited number of nematodes i...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12P19/34
Inventor 梁文举李风平李琪张晓珂
Owner SHENYANG INST OF APPLIED ECOLOGY - CHINESE ACAD OF SCI
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