Method for extracting total DNA (Deoxyribose Nucleic Acid) of mixed chironomidae insect ecdysis on surface of water body

A water surface, mosquito technology, applied in the biological field, to achieve the effect of high purity and high DNA concentration

Pending Publication Date: 2022-06-03
TIANJIN NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The invention overcomes the defects of traditional extraction of eDNA, and provides a method for efficiently extracting mixed chironomid eDNA on the water surface
The purpose of the invention is to solve the method of extracting Chironomidae eDNA more efficiently

Method used

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  • Method for extracting total DNA (Deoxyribose Nucleic Acid) of mixed chironomidae insect ecdysis on surface of water body
  • Method for extracting total DNA (Deoxyribose Nucleic Acid) of mixed chironomidae insect ecdysis on surface of water body
  • Method for extracting total DNA (Deoxyribose Nucleic Acid) of mixed chironomidae insect ecdysis on surface of water body

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

(1): Go to the downwind of rivers and lakes, especially the banks with a little white foam, to collect chironomididae pupa skins.

[0025] (2): soak the collected chironomid pupa skin with 95% alcohol for 5 minutes, soak twice, and then rinse with distilled water three times to remove impurities on the pupa skin.

[0026] (3): soak the pupal skin with physiological saline for two hours to restore the activity of the remaining cells on the pupal skin to facilitate DNA extraction.

[0027] (4): Put the pupa skin into a 37° water bath and keep it at a constant temperature for 2 hours.

[0028] (5): put the treated pupa skin into the mortar, add dissociation solution and fully grind; The composition of described dissociation solution (200 ml) is as follows (W / W):

CTAB 4g final concentration 2%

SDS 1g final concentration 0.5%

0.5M EDTA 8 ml final concentration 0.02M

1M Tris-HCl 20 ml final concentration 0.1M

5M NaCl 40 ml final concentration 1M

β-Mercaptoethanol 0.2 ml f...

Embodiment 2

PCR reaction

(1) PCR reaction system:

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

[0036] (2) PCR reaction conditions:

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

[0037] (3) Spectrophotometric detection of DNA, and electrophoresis detection of PCR products with 1% agarose gel.

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Abstract

The invention discloses a method for extracting total DNA (deoxyribonucleic acid) of mixed chironomidae insect ecdysis on the surface of a water body. The invention mainly investigates the environmental DNA extraction, normal saline treatment and water bath pot preservation time of a sample by adopting pupa skin, and the influence of grinding of pupa skin on DNA extraction during splitting decomposition. The key point is to solve the problems of difficulty in environment DNA extraction, low quality and the like, compared with an electrophoretic band of an improved extraction method, the DNA extracted by the traditional extraction method is fuzzy and darker, while the DNA extracted by the improved extraction method is bright and has no towing phenomenon, which proves that the DNA extracted by the improved method is higher in concentration and higher in purity.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for extracting the total DNA of mixed chironomids molting on the surface of a water body. Background technique [0002] Chironomids belong to the order Diptera, which are numerous and widely distributed. They are distributed in various waters and have strong adaptability to the environment. Its life cycle includes four stages: egg, larva, pupa, and adult. The first three stages are in the water environment, and the adults live out of the water environment. Chironomids lived in water in the first three periods of their life history, and had a strong tolerance to the water environment. They had a huge number in the benthic environment, accounting for more than half of the number of benthic organisms. They were the dominant species of benthic organisms. . There are a lot of chironomids. Some species like to live in relatively clean waters and some like to live in ...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/6806
CPCC12N15/101C12Q1/6806C12Q2523/32C12Q2527/125C12Q2523/308
Inventor 刘文彬龙辰周安陌徐畅翟培闫春财孙小雅
Owner TIANJIN NORMAL UNIVERSITY
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