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Method for extracting DNA of single chironomidae pupal stage ecdysis

A mosquito family, pupal stage technology, applied in DNA preparation, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problem that the total DNA purity and quality cannot meet the requirements of subsequent experiments, and the molecular identification of chironomid pupa skin is difficult to carry out. Unable to extract pupa skin DNA and other problems to prevent interference with extraction

Pending Publication Date: 2020-08-25
TIANJIN NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Compared with larvae and adults, the amount of DNA remaining in the molt of Chironomus pupal stage is very small, and the DNA of the pupal skin cannot be extracted by using the traditional CTAB method 10 times, and there are only traces of DNA
When extracting DNA through traditional CTAB, SDS, NaCl and kit methods, the early DNA cannot be completely separated from the pupal skin and effectively enriched, and the purity and quality of the total DNA obtained cannot meet the requirements of subsequent experiments. The average amount of DNA in the molt of chironomus pupae is 0.003ng / μl~0.020ng / μl, which makes it difficult to carry out molecular identification of chironomus pupal skin

Method used

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  • Method for extracting DNA of single chironomidae pupal stage ecdysis
  • Method for extracting DNA of single chironomidae pupal stage ecdysis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] (1) Preprocessing:

[0052] (11) Take a single molt of chironomus pupal stage, soak it in distilled water at 25°C for 5 minutes, then replace the distilled water and repeat once. The amount of distilled water added is 1-2 cm above the molt of chironomus pupal stage to remove impurities and prevent interference with extraction. .

[0053] (12) After soaking in distilled water, soak in 0.9% NaCl solution again. The amount added is 1-2 cm above the molting stage of chironomus pupal stage. Change it every 1 hour, and change twice for a total of 3 hours. .

[0054] (2) Lysis centrifugation:

[0055] (21) Preparation of lysate: Weigh 2g of CTAB, 0.5g of SDS, 6ml of 0.5M EDTA, 10ml of 1M Tris-HCl, 20ml of 5M NaCl, 1g of PVP and 0.1ml of β-mercaptoethanol, and make up to 100ml with distilled water.

[0056] (22) Put the pretreated chironomus pupae molt in the Ep tube, use dissecting scissors to cut the chironomus pupae molt into small segments with a width less than 2mm, add 1...

Embodiment 2

[0069] Example 2 PCR Amplification of Mitochondrial Cytochrome C Oxidase Subunit I Gene (COI Gene)

[0070] (1) PCR reaction system:

[0071] Total system 50 μl: ddH 2 O 32.4μl, 10ⅹPCR Buffer 5μl, dNTPs (2.5mmol / L) 4μl, universal primer LCO and HCO (10μmol / L) 2μl each, Taq enzyme (500U, 2.5U / ul) 0.6μl, DNA template 4μl.

[0072] (2) PCR reaction conditions:

[0073] Pre-denaturation at 94°C for 5min; denaturation at 94°C for 10s; annealing at 55°C for 30s; extension at 72°C for 60s; 35 cycles; extension at 72°C for 10min; storage at 4°C.

[0074] (3) Configure 1% agarose gel for electrophoresis detection, the results are as follows figure 2 shown.

[0075] figure 2 Among them, the first lane is 8Kp Marker, and the second, third, fourth, and fifth lanes are the PCR results after extracting DNA from Chironomidae pupal molt (Propsilocerus akamusi) of the same species, same size, and same eclosion time After four sets of repetitions, the method of the present invention can...

Embodiment 3

[0077] Embodiment 3 DNA extraction method comparative test

[0078] Verify the difference in the effect of the conventional method and the method described in Example 1 on the extraction of DNA from a single chironomid pupal stage molt.

[0079] According to the traditional CTAB method and the DNA extraction kit sold in the market, according to the steps and the method described in Example 1, take 1 μl and pass through the ddH 2 After O was dissolved, the DNA extracted according to the CTAB method, the kit method and the method described in Example 1 was detected using a NanoDrop 2000 instrument.

[0080] NanoDrop 2000 uses a high-energy xenon lamp. After the light passes through the test sample, part of the light is absorbed. NanoDrop decomposes the light with complex components after passing through the sample into spectral lines, and calculates the absorbance value of the sample to convert it into the concentration of the sample.

[0081] Calculation formula: A=-lg(I / I0)=-...

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Abstract

The invention discloses a method for extracting DNA of a single chironomidae pupal stage ecdysis, and belongs to the technical field of animal molecular biology. The method comprises the following steps of pretreatment, pyrolysis centrifugation, elution, enrichment, enzymolysis, extraction, centrifugation, precipitation, dissolution, amplification and electrophoresis detection. Compared with the prior art, the method has the beneficial effects that distilled water is used for treatment to remove impurities and prevent interference extraction; under the influence of slow permeation of a 0.9% NaCl solution, the cells remaining on the chironomidae pupal stage ecdysis can gradually recover physiological activity in a mild state, and gradual dissociation of cross-linked protein and DNA in the cells is promoted; and the concentration of the chironomidae pupal stage ecdysis DNA extracted by the method can reach 0.908 ng / [mu]l, and the purity can reach 1.89.

Description

technical field [0001] The invention relates to the technical field of animal molecular biology, and more specifically relates to a method for extracting the molt DNA of a single Chironomidae pupal stage. Background technique [0002] Chironomids are insects of the order Diptera Longhorn. Due to the diversity of ecological requirements and living habits among different species, the differences in sensitivity to environmental factors make them excellent indicators for biological monitoring of water environments. Some species can carry pathogens to cause asthma, cholera, dermatitis and other diseases, endangering human health, and some species are important agricultural and fishery pests, harming rice, water shield and other crops. [0003] The pupal stage of chironomus is a developing adult larva with a double-layer body wall. It connects the two stages of larva and adult, and has some similar structures and traits with the two stages, as well as unique morphological charact...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/101C12Q2523/308C12Q2523/32C12Q2521/327C12Q2521/537
Inventor 刘文彬闫春财孙小雅龙辰
Owner TIANJIN NORMAL UNIVERSITY
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