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Method for efficiently extracting midge larva DNA (deoxyribonucleic acid)

A high-efficiency technology for chironomus larvae, applied in DNA preparation, recombinant DNA technology, etc., can solve problems such as poor results, and achieve low cost, good quality, and high success rate

Inactive Publication Date: 2015-12-02
TIANJIN NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Both the traditional method and the kit method have their own advantages and disadvantages, but in the extraction of chironomid larval DNA, both are not effective

Method used

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  • Method for efficiently extracting midge larva DNA (deoxyribonucleic acid)
  • Method for efficiently extracting midge larva DNA (deoxyribonucleic acid)
  • Method for efficiently extracting midge larva DNA (deoxyribonucleic acid)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: DNA extraction from chironomid larvae

[0042] Preparation of lysate (100ml):

[0043] CTAB2g final concentration 2%

[0044] SDS0.5g final concentration 0.5%

[0045] 0.5 MEDTA 6ml final concentration 0.03M

[0046] 1MTris-HCl10ml final concentration 0.1M

[0047] 5MNaCl20ml final concentration 1M

[0048] PVP1g final concentration 1%

[0049] β-mercaptoethanol 0.1ml final concentration 0.1%

[0050] Make up to 100ml with distilled water.

[0051] (1) Pretreatment:

[0052] a. Take a single chironomid larva and mount the head shell, use a dissecting needle to pull out all the intestines and discard them as much as possible to prevent the digestion in the intestine from interfering with subsequent experiments.

[0053] b. Soak the insect body with distilled water, and change the distilled water every 5 minutes for a total of 15 minutes to remove impurities on the insect body. Finally, use absorbent paper to absorb the distilled water on the surface of the insect body.

[0054...

Embodiment 2

[0063] PCR amplification of mitochondrial cytochrome C oxidase subunit I gene (COI gene)

[0064] (1) PCR reaction system:

[0065] Total system 25ul: ddH 2 O16.2ul, 10ⅹPCRBuffer 2.5ul, dNTPs (2.5mmol / L) 2ul, universal primer LCO and HCO (10umol / L) each 1ul, Taq enzyme (500U, 2.5U / ul) 0.3ul, DNA template 2ul.

[0066] (2) PCR reaction conditions:

[0067] Pre-denaturation at 94°C for 5min; denaturation at 94°C for 40s; annealing at 56°C for 40s; extension at 72°C for 1 min; 30 cycles; extension at 72°C for 5 min; storage at 4°C.

[0068] (3) Configure 1% agarose gel for electrophoresis detection, see attached picture.

[0069] Explanation of results:

[0070] (1) The first lane of the electrophoresis graph is 8KpMarker, and the second lanes 2, 3, 4, and 5 are the PCR results of a single red chironomid larva after DNA extraction.

[0071] (2) The COI gene is about 698bp, between the first and second bands of the Marker, and the result is in line with expectations.

Embodiment 3

[0073] Comparative Test

[0074]

[0075] Conclusion: Compared with conventional methods, the method of the present invention has higher DNA concentration and higher purity.

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Abstract

The invention discloses a method for efficiently extracting midge larva DNA (deoxyribonucleic acid), which comprises the following steps: taking a single midge larva, pulling out and discarding all intestinal tracts with a dissecting needle to the greatest extent, immersing the larva in distilled water, putting the obtained larva in a 1.5ml Ep tube, adding a lysis solution, and sufficiently grinding the larva with a grinding rod; adding 20 mu l of proteinase K and 15 mu l of ribonuclease, uniformly mixing, putting in a 56-DEG C water bath, and carrying out digestive treatment over night; extracting with chloroform and isoamyl alcohol twice; adding 300 mu l of saturated phenol and 300 mu l of chloroform; extracting with isoamyl alcohol once; finally, adding 0.5 volume of isopropanol ice, and precipitating; and washing with 70% ethanol, drying at 37 DEG C in a drying oven, and adding 30 mu ldd of H2O (37 DEG C) to dissolve the DNA. The method can obtain the midge larva high-quality DNA, and the midge larva high-quality DNA can be directly used in PCR (polymerase chain reaction) to amplify the COI gene.

Description

[0001] This invention has been awarded by Tianjin Natural Science Foundation (14JCQNJC14600), National Natural Science Foundation of China (31101653, 31272284), Tianjin University Science and Technology Development Fund (20090608), Tianjin Normal University Introduced Talent Fund (5RL104), and College Student Innovation and Entrepreneurship Training Program (201410065046) ) And Tianjin Excellent Young Teachers Funding Program (2013). Technical field [0002] The invention belongs to the technical field of animal molecular biology, and particularly relates to a method for efficiently extracting DNA from chironomid larvae. Background technique [0003] Chironomids belong to the suborder Diptera: Nematocera (Diptera: Nematocera) in the class of insects. The adult body shape is similar to that of mosquitoes. Because of their mouthparts degeneration, they are called "non-biting midges". It is named because the forefoot keeps swinging when the adult is stationary, so it is called the mi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 闫春财闫娇戈昕宇郭琴
Owner TIANJIN NORMAL UNIVERSITY
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