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Method for extracting high-quality DNA (deoxyribonucleic acid) in dry leaves of soybeans suitable for transgenic detection

An extraction method and technology of dried leaves, which are applied in the field of life sciences, can solve the problems that DNA contains a lot of salt ions and impurities, cannot remove pigment substances, and cannot effectively remove polysaccharides, etc., to achieve simple extraction, obvious specific products, and transparent color Effect

Inactive Publication Date: 2013-08-14
QILU UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional SDS method cannot effectively remove polysaccharides and other substances, and the extracted DNA contains more salt ions and impurities
Compared with the SDS method, the traditional CTAB method removes polysaccharides more cleanly, but the DNA yield is relatively low and cannot remove a large amount of pigment substances contained in dry old leaves, and the obtained DNA is not pure

Method used

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  • Method for extracting high-quality DNA (deoxyribonucleic acid) in dry leaves of soybeans suitable for transgenic detection
  • Method for extracting high-quality DNA (deoxyribonucleic acid) in dry leaves of soybeans suitable for transgenic detection
  • Method for extracting high-quality DNA (deoxyribonucleic acid) in dry leaves of soybeans suitable for transgenic detection

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Example 1: Extraction of soybean dry leaf genome total DNA

[0032] (1) The method steps of the present invention are as follows:

[0033] ①Take about 0.04g of dried soybean leaves, put them into a 1.5mL centrifuge tube, add 200μL of Buffer I (0.2mol / L tris-HCl, 0.05mol / L EDTA, 1mol / L NaCl, pH8.0), soak for 10 minutes , heated in a water bath at 60°C, cut the macerated leaves with small scissors, and obtained about 0.04g of foam;

[0034] ②Add 1.2mL Buffer II (the mass ratio of sterile water to β-mercaptoethanol is 40:1), mix upside down for 5 minutes, centrifuge at 10000r / min for 5 minutes, discard the supernatant, repeat this step 2-3 times, Until the supernatant is clarified;

[0035] ③ Take the precipitate, add 600 μL 2×CTAB extract (containing 2% PVP, 1.4mol / L NaCl), 3% β-mercaptoethanol, 2 μL proteinase K, shake well, and bathe in 65°C water for 60 minutes. Shake it upside down every 10 minutes to get a mixture;

[0036] ④ Add the same volume of phenol: chloro...

Embodiment 2

[0049] Example 2: Extraction of soybean dry leaf genome total DNA

[0050] ①Take about 0.04g of dried soybean leaves, put them into a 1.5mL centrifuge tube, add 200μL of Buffer I (0.2mol / L tris-HCl, 0.05mol / L EDTA, 1mol / L NaCl, pH8.0), soak for 15 minutes , heated in a water bath at 40°C, and chopped the soaked leaves with small scissors;

[0051] ②Add 1.2mL Buffer II (the mass ratio of sterile water to β-mercaptoethanol is 40:1), mix upside down for 5 minutes, centrifuge at 10000r / min for 5 minutes, discard the supernatant, repeat this step 2-3 times, Until the supernatant is clarified;

[0052] ③ Take the precipitate, add 600 μL 2×CTAB extract (containing 2% PVP, 1.4mol / L NaCl), 3% β-mercaptoethanol, 2 μL proteinase K, shake well, and bathe in 65°C water for 40 minutes. Shake up and down every 10 minutes;

[0053] ④ Add an equal volume of phenol: chloroform: isoamyl alcohol (volume ratio 25:24:1) mixture, mix upside down for 10 minutes, and centrifuge at 12000r / min for 10...

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Abstract

The invention discloses a method for extracting high-quality DNA (deoxyribonucleic acid) in dry leaves of soybeans suitable for transgenic detection. According to the method, extracted genomes are clear in DNA strips, high in purity and low in impurity content, and meet requirements for molecular biology experiments such as PCR (polymerase chain reaction), enzyme digestion, molecular cloning, sequencing and the like required by the transgenic detection. The method comprises the following steps of (1) soaking the dry leaves of the soybeans with Buffer I, heating at 40-60 DEG C, and cutting into powder; (2) adding Buffer II, shaking up, centrifuging, removing supernatant, repeating the operations until the supernatant is clarified, and taking precipitates; (3) adding a CTAB (cetyl trimethyl ammonium bromide) extracting solution, beta-mercaptoethanol of which the mass concentration is 3% and protease K, and heating for 40-60 minutes at 55-65 DEG C; (4) adding a mixed solution of phenol, chloroform and isoamyl alcohol equal in volume, uniformly mixing, centrifuging, and taking the supernatant; (5) adding a mixed solution of chloroform and isoamyl alcohol equal in volume, uniformly mixing, centrifuging, and taking the supernatant; and (6) adding pre-cooled isopropyl alcohol 0.8 times in volume, standing for 10-30 minutes at -20 DEG C, centrifuging, removing the supernatant, washing, airing, and dissolving in a TE buffering solution.

Description

technical field [0001] The invention relates to a DNA extraction method, in particular to a method for extracting high-quality DNA from dried soybean leaves suitable for transgene detection. Belongs to the field of life sciences. Background technique [0002] DNA extraction is the basis of biotechnology research and is crucial for subsequent molecular biology experiments. Improving the integrity and purity of genomic DNA is the key to high-quality DNA extraction, and it is also related to molecular hybridization, molecular cloning, transgenic detection, The success or failure of follow-up studies such as genetic diversity analysis. The quality of genomic DNA extraction is also closely related to the type and location of the material, and the extraction methods for different materials are also different. Obtaining high-quality DNA that can be used for PCR amplification from dried soybean leaves is of great significance for high-throughput sample analysis. The general conve...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/68
Inventor 王存芳许晓丹刘畅史永翠
Owner QILU UNIV OF TECH
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