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Method for extracting plant deoxyribonucleic acid (DNA) and special kit thereof

A kit and plant technology, applied in the field of extracting plant DNA, can solve the problems of limited use and poor extraction effect, and achieve the effects of good versatility, low cost and high yield

Inactive Publication Date: 2014-02-05
INST OF BOTANY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional CTAB method is the most widely used DNA extraction method at present, but due to differences in the chemical composition and tissue structure of plant materials, the extraction effect of the traditional CTAB method is sometimes not good, and its use is subject to certain restrictions

Method used

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  • Method for extracting plant deoxyribonucleic acid (DNA) and special kit thereof
  • Method for extracting plant deoxyribonucleic acid (DNA) and special kit thereof
  • Method for extracting plant deoxyribonucleic acid (DNA) and special kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1, the method for extracting plant DNA of the present invention (hereinafter referred to as mCTAB method)

[0043] 1. Weigh 20mg of plant dry material, add quartz sand, grind it into powder, and transfer the powder to a 2.0mL centrifuge tube.

[0044] 2. Add 1mL of pre-cooled buffer A, mix well and then ice-bath for 15min; invert and mix 2-3 times during the ice-bath. Centrifuge at 7000×g for 10 min and discard the supernatant.

[0045] 3. Repeat step 2 until the supernatant is not viscous.

[0046] 4. Add 0.7mL buffer B (Table 3), mix well and then bathe in 65°C water bath for 90-120min, invert and mix several times during the water bath process. Centrifuge at 10000×g for 10min, and aspirate the supernatant into a new 2.0mL centrifuge tube.

[0047] 5. Add 0.7 mL of chloroform isoamyl alcohol solution (chloroform: isoamyl alcohol = 24:1), invert and mix for 10 min. Centrifuge at 10000×g for 10min, and aspirate the supernatant into a new 1.5mL centrifuge ...

Embodiment 2

[0059] The method is basically the same as in Example 1, the differences are as follows:

[0060] Buffer A is composed of solute and solvent; the solute and its final concentration in buffer A are as follows: disodium edetate 4mmol L -1 , NaCl 0.2M, polyvinylpyrrolidone 1g / 100ml; 1M Tris-HCl buffer solution with pH 8.0 8ml / 100ml; the solvent is water.

[0061] Buffer B is composed of solute and solvent: solute and its final concentration in buffer B are as follows: 1M Tris-HCl buffer solution with pH=8.0 8ml / 100ml, disodium edetate 20mmol L -1 , NaCl 1.2M, CTAB 2.5g / 100ml, sodium metabisulfite 0.5g / 100ml, sodium ascorbate 0.5g / 100ml, polyvinylpyrrolidone 1g / 100ml, β-mercaptoethanol 80ul / 100ml; the solvent is water.

[0062] The time of the ice bath was 10 minutes; the condition of the water bath was 90 minutes at 60°C.

Embodiment 3

[0064] The method is basically the same as in Example 1, the differences are as follows:

[0065] Buffer A is composed of solute and solvent; the solute and its final concentration in buffer A are as follows: EDTA disodium 6mmol L -1 , NaCl 0.3M, polyvinylpyrrolidone 3g / 100ml; 1M Tris-HCl buffer solution with pH 8.0 12ml / 100ml; the solvent is water.

[0066] Buffer B is composed of solute and solvent: solute and its final concentration in buffer B are as follows: 1M Tris-HCl buffer solution with pH=8.0 12ml / 100ml, disodium edetate 30mmol L -1 , NaCl 1.6M, CTAB 3.5g / 100ml, sodium metabisulfite 1.5g / 100ml, sodium ascorbate 1.5g / 100ml, polyvinylpyrrolidone 3g / 100ml, β-mercaptoethanol 120ul / 100ml; the solvent is water.

[0067] The time of the ice bath was 20 minutes; the condition of the water bath was 120 minutes at 70°C.

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Abstract

The invention aims to provide a method for extracting plant deoxyribonucleic acid (DNA) and a special kit thereof. The special kit comprises a buffer solution A which consists of solute and solvent, wherein the final concentrations of the solute and the solute in the buffer solution A are as follows: 4-6mmol.L<-1> of ethylene diamine tetraacetic acid, 0.2-0.3M of NaCl, 1-3g / 100ml of polyvinyl pyrrolidone, 1M of Tris-HCl of which the pH value is 8.0 and 8-12ml / 100ml of buffer solution; and the solvent is water. The plant DNA extracted by a mCTAB method has the advantages of being high in yield, better in quality, high in PCR (polymerase chain reaction) amplification success rate, low in cost and good in generality, can satisfy the requirements of extracting DNA in molecular biological studies and is especially suitable for researchers with insufficient cost of scientific research and extraction of the plant DNA on a large scale.

Description

technical field [0001] The invention relates to a method for extracting plant DNA and a special kit thereof. Background technique [0002] DNA is the basic genetic material of plants and the carrier of plant genetic information. A certain amount of high-quality DNA samples are the basis for molecular biology research such as restriction enzyme digestion, PCR amplification, molecular hybridization, genetic polymorphism analysis, and genomics. Therefore, how to obtain a certain amount and high-quality DNA samples is extremely important. [0003] Plant cells have cell walls, which contain more secondary metabolites such as polysaccharides, and the types and contents of secondary metabolites in different plants vary greatly, and sometimes the types and contents of secondary metabolites in different organs or tissues of the same plant are also different. Similarly, it is difficult to obtain high-quality DNA. A DNA extraction method optimized for one particular species may not ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10
Inventor 周世良李金璐于婧王硕
Owner INST OF BOTANY CHINESE ACAD OF SCI
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