Damage-free cold extraction method for genome DNA of small insects

A genomic and cold extraction technology, applied in the field of molecular biology, can solve the problems of insufficient DNA release, genomic DNA degradation, affecting DNA quality, etc., to reduce DNA degradation, enhance digestion, and improve yield.

Inactive Publication Date: 2015-05-20
ZHENGZHOU TOBACCO RES INST OF CNTC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still some shortcomings in this method: (1) the digestion by proteinase K alone is not enough to fully release the DNA in the insect body, especially some insects whose body surface is wrapped by chitin shell; (2) the digestion by proteinase K Finally, the protease is not inactivated by high temperature, and the residual enzyme will have a certain impact on subsequent experiments; (3) Long-term incubation at 56°C will degrade genomic DNA and affect DNA quality

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  • Damage-free cold extraction method for genome DNA of small insects
  • Damage-free cold extraction method for genome DNA of small insects
  • Damage-free cold extraction method for genome DNA of small insects

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Genomic DNA of Hymenoptera ants (sample 1) was extracted.

[0030] 1 Preparation of extraction buffer

[0031] Prepare 400 μL extraction buffer: 40 μL 10×PCR Buffer Ⅱ, 1.8 μL NP40, 1.8 μL Tween20, 12 μL proteinase K (20 mg / mL), 344.4 μL ddH 2 O.

[0032] 2 Pretreatment of worms

[0033] First carefully clean the impurities on the surface of the insect body with 95% alcohol, then wash it three times with distilled water, and finally use absorbent paper to dry the water on the surface of the insect body.

[0034] 3 Extraction of genomic DNA

[0035] (1) Put the ants into a centrifuge tube, add 300 μL of extraction buffer, bathe in water at 50°C for 1 hour, and mix by gently inverting every 15 minutes;

[0036] (2) Store at -20°C for 15 hours, and cold extract genomic DNA;

[0037] (3) Add 9 μL of proteinase K (20 mg / mL), bathe in 50°C water for 1 hour, and mix by gently inverting every 15 minutes;

[0038] (4) Water bath at 95°C for 10 minutes to inactivate proteina...

Embodiment 2

[0046] Genomic DNA of Homoptera leafhopper (sample 2) was extracted.

[0047] 1 Preparation of extraction buffer

[0048] Prepare 300 μL extraction buffer: 30 μL 10×PCR Buffer Ⅱ, 1.35 μL NP40, 1.35 μL Tween20, 9 μL proteinase K (20 mg / mL), 258.3 μL ddH 2 O.

[0049] 2 Pretreatment of worm bodies (same as Example 1)

[0050] 3 Extraction of genomic DNA

[0051] (1) Put the leafhopper into a centrifuge tube, add 200 μL of extraction buffer, bathe in water at 50°C for 1 hour, and mix by gently inverting every 15 minutes;

[0052] (2) Store at -20°C for 15 hours, and cold extract genomic DNA;

[0053] (3) Add 6 μL of proteinase K (20 mg / mL), bathe in 50°C water for 1 hour, and mix by gently inverting every 15 minutes;

[0054] (4) Water bath at 95°C for 10 minutes to inactivate proteinase K;

[0055] (5) Take the leafhopper out of the centrifuge tube with sterile tweezers, and put it in 95% alcohol for storage. The solution in the centrifuge tube contains the genomic DNA of...

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Abstract

The invention discloses a damage-free cold extraction method for genome DNA of small insects. The method comprises the following several steps: (1) preparing a genome DNA extraction buffer solution; (2) cleaning impurities on the surfaces of the insects by using 95% alcohol, and cleaning by using distilled water; (3) adding the insects into a centrifuge tube, adding the extraction buffer solution which is 3-6 times that of the volume of the insect body into a water bath at the temperature of 50+ / -5 DEG C; (4) standing at the temperature of 20 DEG C below zero, and performing cold extraction on the genome DNA; (5) adding protease K so as to reach the final concentration of 0.6mg / mL, and reacting in the water bath at the temperature of 50+ / -5 DEG C; (6) inactivating the protease K at the temperature of 95 DEG C; and (7) taking the insects out of the centrifuge tube, wherein the genome DNA of the insects is contained in the solution in the tube. According to the extraction method, the integrity of the insect body can be maintained, and due to cold extraction at the temperature of 20 DEG C below zero, the genome DNA of the insects can be fully released from the insect bodies under low-temperature conditions, degradation of the genome DNA is avoided, and the quality of the genome DNA is improved.

Description

technical field [0001] The invention belongs to the field of molecular biology and relates to a molecular biology method for non-damaging cold extraction of small insect genome DNA. Background technique [0002] The genomic DNA of insects is of great significance to the study of taxonomy and identification, phylogenetic evolution, and species diversity of insects. High-quality DNA is the prerequisite for related research. The traditional method of extracting insect genome DNA needs to crush and grind the insect body first, and then undergo protease digestion and organic solvent extraction to finally obtain genome DNA. These methods will cause permanent damage to insect samples and are not conducive to preserving the integrity of samples, especially some rare insect samples, which will have a certain impact on subsequent morphological classification and identification. At the same time, traditional extraction methods need to use organic solvents such as phenol, chloroform, a...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 魏攀李锋罗朝鹏王燃魏春阳王中武明珠张剑锋金立锋杨军林福呈
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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