Third-generation sequencing phytoplankton genomic DNA extraction method and application thereof

A zooplankton and extraction method technology, applied in the field of genetic engineering, can solve the problems of inability to meet the requirements of building a library, costing huge manpower and material resources, etc., and achieve the effects of complete fragmentation, improved purity, and high purity.

Active Publication Date: 2019-02-15
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the process of extracting Cladocera genomic DNA with conventional kits, we found that the genomic DNA solutions of some transparent species (such as Diaphanosoma, Leptodora and Cercopagidae) contained a large amount of pigment, A260/230 is always l

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  • Third-generation sequencing phytoplankton genomic DNA extraction method and application thereof
  • Third-generation sequencing phytoplankton genomic DNA extraction method and application thereof
  • Third-generation sequencing phytoplankton genomic DNA extraction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] (1) Individual collection of Diaphanosoma dubium, the steps are as follows:

[0064] The daphnia spp. fuzzy uses fresh living body, and this species can achieve absolute dominance in summer (June to August) in some water bodies. Therefore, in this experiment, water samples of zooplankton were collected from a lake on a university campus in Guangzhou by using a 120 μm trawl net. In the laboratory, the water samples containing zooplankton passed through 600 μm and 250 μm mesh screens in turn. Immediately transfer the zooplankton above the 250μm mesh sieve to a 1L beaker filled with drinking purified water, in which 100u / L penicillin and 100μg / L streptomycin were added in advance to reduce pollution. After 5 to 6 hours, manually select the required fuzzy Daphnia spp. under incandescent light. In order to ensure the purity of Daphnia chevrosum, the selection was repeated 2 to 3 times, and the selected individuals were put into pure water containing the same antibiotic conc...

Embodiment 2

[0078] (1) Individual collection of three kinds of Cladocera, the steps are as follows:

[0079] Three species of Cladocera (Diaphanosoma dubium, D.dubium), Leptodora richard (L.richard) and Bythotrephes longimanus (Bythotrephes longimanus , B.longimanus)) conducted an experimental comparison; where:

[0080] The daphnia spp. fuzzy uses fresh living body, and this species can achieve absolute dominance in summer (June to August) in some water bodies. Therefore, in this experiment, water samples of zooplankton were collected from a lake on a university campus in Guangzhou by using a 120 μm trawl net. In the laboratory, the water samples containing zooplankton passed through 600 μm and 250 μm mesh screens in turn. Immediately transfer the zooplankton above the 250μm mesh sieve to a 1L beaker filled with drinking purified water, in which 100u / L penicillin and 100μg / L streptomycin were added in advance to reduce pollution. After 5 to 6 hours, manually select the required fuzzy D...

Embodiment 3

[0097] (1) Individual collection of zooplankton, the steps are as follows:

[0098] For the zooplankton, all the fresh live zooplankton collected in the water body at one time were used. In this experiment, zooplankton water samples were collected from a lake on a university campus in Guangzhou by using a 120 μm trawl net. In the laboratory, the water samples containing zooplankton were passed through a 600 μm mesh sieve to remove impurities. Immediately transfer the zooplankton in the water sample to a 1L beaker filled with drinking purified water, in which 100u / L penicillin and 100μg / L streptomycin were added in advance to reduce pollution. After 5-6 hours, the zooplankton was collected again with a 120 μm mesh sieve, and put into purified water containing the same antibiotic concentration to obtain fresh individual zooplankton.

[0099] (2) Zooplankton genome DNA extraction, the steps are as follows:

[0100] 1) Take the kit ( The buffer solution GA in the company's mar...

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Abstract

The invention discloses a third-generation sequencing phytoplankton genomic DNA extraction method and application thereof. The method comprises the following steps: (1) adding 18% to 20% (V/V) beta-mercaptoethanol to a buffer GA, then adding zooplankton, and grinding to obtain a tissue buffer solution; (2) adding protease PK to the tissue buffer for digestion, then adding RNase A, allowing standing still, centrifuging to obtain supernatant I, adding a solvent, centrifuging to obtain supernatant II, finally adding a buffer GB, mixing uniformly, and adding anhydrous ethanol to obtain a mixed solution; (3) adding the mixed solution to an adsorbent column CB3, rinsing and eluting to obtain phytoplankton genomic DNA. The method disclosed by the invention is simple, easy to operate, and capableof effectively removing pigments, and obtained phytoplankton genomic DNA fragments are complete, high in purity, and high in concentration, thereby being capable of providing the assurance for smoothimplementation of the third-generation sequencing of zooplanktons, especially cladocerans.

Description

technical field [0001] The invention belongs to the field of genetic engineering, in particular to a third-generation sequencing zooplankton genome DNA extraction method and application. Background technique [0002] Cladocerans belong to an ancient group of crustaceans, according to the fossil record and DNA data. Their zoogeographical features are: some faunal complexes and taxa show bipolar distribution patterns; some species have wide ranges and others have narrow ranges; isolated populations and relatively concentrated in the temperate-subtropical regions of the two hemispheres native species. These are beneficial for us to analyze the historical reasons for the formation of cladocera fauna by comparing with plants, invertebrates and vertebrates. Therefore, the genome sequencing of certain species of Cladocera is beneficial for us to carry out their population evolution and functional gene analysis, discuss the genetic structure of the species and the genetic structur...

Claims

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Application Information

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IPC IPC(8): C12Q1/6806C12Q1/6869C12N15/10
CPCC12N15/1003C12Q1/6806C12Q1/6869C12Q2535/122
Inventor 韩博平刘平徐少林
Owner JINAN UNIVERSITY
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