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Method of extracting tussah blood DNA

An extraction method, the technology of tussah silkworm, applied in the field of molecular biology, can solve the problems affecting the extraction of genomic DNA, time-consuming and labor-intensive DNA extraction, and easy inhibition of Tag enzyme activity, etc., to achieve scientific and reasonable extraction steps, low cost, and strong practicability Effect

Inactive Publication Date: 2017-05-10
JILIN SERICULTURE SCI RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The DNA extraction of these sample materials either requires liquid nitrogen for grinding or long-term digestion, and the entire DNA extraction is time-consuming and labor-intensive.
Moreover, these samples contain a large amount of impurities such as fat and protein

Method used

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  • Method of extracting tussah blood DNA

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Experimental program
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Effect test

Embodiment 1

[0033] Embodiment 1: a kind of tussah silkworm blood DNA extraction method of embodiment 1, comprising:

[0034] 1. Solution configuration

[0035] (1) Extraction buffer: Each 1L of extraction buffer contains 10 mmol of Tris-HCl, 10 mmol of EDTA, 100 mmol of NaCl, 2% of SDS, and 0.039 mol of DTT, and the balance is water, which uses DNase / RNase-Free deionized water, the pH of the extraction buffer = 8.0;

[0036] (2) Chloroform-isoamyl alcohol: a mixed solution of chloroform-isoamyl alcohol with a volume ratio of 24:1;

[0037] (3) 70% ethanol solution: ethanol, DNase / RNase-Free deionized water mixed solution with a volume ratio of 7:3;

[0038] (4) TE buffer: Each 1L of the TE buffer contains 10 mmol of Tris-HCl and 1 mmol of EDTA, and the balance is water. The water uses DNase / RNase-Free deionized water, and the pH of the TE buffer is = 8.0;

[0039] 2. Extraction of tussah silkworm DNA

[0040] 1) Tussah silkworm blood treatment: take 1.3ml blood of tussah silkworm 4~...

Embodiment 2

[0047] Embodiment 2: a kind of tussah silkworm blood DNA extraction method of embodiment 2, comprising:

[0048] One, solution configuration is identical with embodiment 1;

[0049] 2. Extraction of tussah silkworm DNA

[0050] 1) Tussah silkworm blood treatment: take 1.3ml blood of tussah silkworm 4~5 instar larvae, put it into a centrifuge, centrifuge at 12000rpm for 8min, discard the supernatant, and get a white precipitate;

[0051] 2) Add 1.3ml of extraction buffer to the white precipitate, shake and mix well, and dissolve the precipitate in the extraction buffer to obtain the mixture I;

[0052] 3) Add 0.5 mg of proteinase K and 0.05 mg of ribonuclease A to the mixture I, and place it at 55°C for 1.5 hours of constant temperature shaking, so that the final concentration of proteinase K is 0.38 mg / ml and the final concentration of ribonuclease A is 0.038 mg / ml, to obtain the lysis mixture II;

[0053] 4) Transfer 0.6ml of lysis mixture II into a new 1.5ml centrifuge tu...

Embodiment 3

[0057] Embodiment 3: a kind of tussah silkworm blood DNA extraction method of embodiment 3, comprising:

[0058] One, solution configuration is identical with embodiment 1;

[0059] 2. Extraction of tussah silkworm DNA

[0060] 1) Tussah silkworm blood treatment: take 1.3ml blood of tussah silkworm 4~5 instar larvae, put it into a centrifuge, centrifuge at 12000rpm for 8min, discard the supernatant, and get a white precipitate;

[0061] 2) Add 1.3ml of extraction buffer to the white precipitate, shake and mix well, and dissolve the precipitate in the extraction buffer to obtain the mixture I;

[0062] 3) Add 0.5 mg of proteinase K and 0.05 mg of ribonuclease A to the mixture I, place it at 60°C for 2 hours, and shake at a constant temperature, so that the final concentration of proteinase K is 0.38 mg / ml and the final concentration of ribonuclease A is 0.038 mg / ml to obtain the lysis mixture II;

[0063] 4) Transfer 0.6ml of lysis mixture II into a new 1.5ml centrifuge tub...

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Abstract

The invention relates to the technical field of molecular biology, in particular to a method of extracting tussah blood DNA, comprising the specific steps of treating tussah blood to obtain white precipitate; adding extracting buffer solution to the white precipitate to obtain mixed liquid I; adding protease K and ribonuclease A to the mixed liquid I to obtain mixed lysate II; adding chloroform-isoamyl alcohol mixed liquid to the mixed lysate II to obtain first supernate; adding absolute ethyl alcohol to the first supernate to obtain colorless clear colloidal tussah DNA; dissolving the air-dried colorless clear colloidal tussah DNA fully in 0.05 ml of TE buffer solution to obtain colorless clear tussah DNA solution. The method of the invention is scientific and reasonable, high in practicality, good in convenience, low in time and labor consumption, simple to perform, high in efficiency, and low in cost, and DNA extracted by using the method is effective and good in quality.

Description

technical field [0001] The invention relates to the technical field of molecular biology, and relates to a method for extracting tussah silkworm blood DNA. Background technique [0002] DNA (Deoxyribonucleic acid), also known as deoxyribonucleic acid, is an important component of living organisms and the material basis of life science research. High-quality genomic DNA is a necessary prerequisite for downstream technologies such as molecular markers, gene cloning, and gene expression research. Therefore, the separation and purification of DNA has become an important link in molecular biology research. [0003] Tussah silkworm (Antheraea pernyi) is a silk-producing insect belonging to the family Lepidoptera and is a unique insect resource with important economic value in my country. In recent years, molecular biology techniques have developed rapidly in the research of tussah silkworms and other insects. The extraction of genomic DNA is the basis of molecular biology experi...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1003
Inventor 张洋李凤芹金英
Owner JILIN SERICULTURE SCI RES INST