Improved CTAB (cetyltrimethylammonium bromide) method for extracting genomic DNA of cotton

A technology of cotton genome and extraction solution, which is applied in the field of DNA extraction, can solve the problems of long time-consuming RNA removal and cumbersome steps, and achieve the effects of reducing DNA extraction cost, high DNA quality, and shortening extraction time

Pending Publication Date: 2018-02-16
COTTON RES INST SHANXI ACAD OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide an improved CTAB method for extracting cotton genomic DNA, which is used to solve the technical problems of cumbersome technical steps for DNA extraction and long time-consuming RNA removal.

Method used

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  • Improved CTAB (cetyltrimethylammonium bromide) method for extracting genomic DNA of cotton
  • Improved CTAB (cetyltrimethylammonium bromide) method for extracting genomic DNA of cotton
  • Improved CTAB (cetyltrimethylammonium bromide) method for extracting genomic DNA of cotton

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Genomic DNA Extraction of Upland Cotton Leaves

[0041] A kind of improved CTAB method extracts cotton genome DNA, comprises the following steps:

[0042] (1) Take the leaves of upland cotton as the experimental material, take 100~200 mg sample and put it into a 2.0mL centrifuge tube, grind it into powder with liquid nitrogen, then add 1000 μL of CTAB buffer solution, shake well, and place in a 65°C water bath React in medium for 40-60 minutes, and gently shake every 10 minutes to obtain DNA-containing extract I. The powder is mixed with CTAB buffer according to m:v ratio of 1-2:10.

[0043] The CTAB buffer contains: 1.4 M NaCl (sodium chloride), 0.02 M EDTA (pH8.0), 0.1 MTris-HCl (pH7.5), 2% (w / v) cetyltrimethyl Ammonium bromide (CTAB), 2% (w / v) PVP-K30, 0.1% (w / v) DIECA, 0.2% (v / v) β-mercaptoethanol, the solvent is deionized water, where β-mercapto Ethanol is added now.

[0044] (2) Put the extraction solution I centrifuge tube containing the DNA in ice ...

Embodiment 2

[0051] Embodiment 2 Determination of RNase A action temperature

[0052] Several copies of upland cotton leaf materials were prepared, and the genomic DNA of upland cotton leaves was extracted by the method in Example 1. Among them, in step (2), place the centrifuge tube containing the DNA extraction solution I in ice to cool down, and then add 1% (v / v) RNase A (purchased from Treasure Biological Company, the specification is 10mg / ml) , and then digested at 22°C, 27°C, 32°C, 37°C, and 65°C for 2 hours in a water bath, respectively.

[0053] Configure 0.8% agarose gel, and use 1×TAE electrophoresis buffer to perform electrophoresis detection on the extracted genomic total DNA. The composition of the 1×TAE electrophoresis buffer is as follows: 0.04mol / L Tris-acetic acid, 0.001mol / L EDTA. Genomic DNA of upland cotton leaves extracted with the kit (Tiangen DP320) was used as a control, and agarose gel electrophoresis was performed together with the genomic DNA of upland cotton le...

Embodiment 3

[0054] Example 3 Determination of RNase A action time

[0055] Several copies of upland cotton leaf materials were prepared, and the genomic DNA of upland cotton leaves was extracted by the method in Example 1. Among them, in step (2), place the centrifuge tube containing the DNA extraction solution I in ice to cool down, add 1% (v / v) RNase A, and then digest it in a water bath at 37°C for 2 hours, 1h, 0.5h, 15min, 7.5min.

[0056] Genomic DNA of upland cotton leaves extracted with the kit (Tiangen DP320) was used as a control, and agarose gel electrophoresis was performed together with the genomic DNA of upland cotton leaves obtained under the action of the above-mentioned RNase at different digestion times. The results are shown in figure 2 . Such as figure 2 As shown, 1% (v / v) concentration of RNase A reacted in a water bath at 37°C for 7.5 minutes is sufficient to fully digest the RNA in the genome.

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Abstract

The invention discloses an improved CTAB (cetyltrimethylammonium bromide) method for extracting genomic DNA of cotton. The method comprises the following steps: (1) grinding liquid nitrogen into powder, adding a CTAB buffer solution, and shaking the mixed solution in a water bath at 65 DEG C to obtain an extract solution I containing DNA; (2) performing cooling, adding RNase A, and performing a water bath at 37 DEG C; (3) adding equal volume of a solution A, uniformly mixing the solutions and performing centrifugation to obtain a supernatant I; (4) continuously adding equal volume of the solution A, uniformly mixing the solutions and performing centrifugation to obtain a supernatant II; (5) adding NaAc, then adding absolute ethyl alcohol, and performing shaking horizontally to form flocculent precipitates; (6) performing centrifugation, removing the supernatants, washing the precipitates, removing absolute ethyl alcohol, and performing drying to obtain precipitated DNA; (7) dissolvingthe precipitated DNA and storing the DNA in a refrigerator at subzero 20 DEG C for standby application. The method has the advantages that procedures for extracting genomic DNA of cotton leaves with the CTAB method are simplified, experimental time is shortened greatly, DNA extraction cost is reduced, RNA eliminating effect is improved, and the method is applicable to large-scale extraction of genomic DNA of the cotton leaves.

Description

technical field [0001] The invention relates to the technical field of DNA extraction, in particular to an improved CTAB method for extracting cotton genome DNA. Background technique [0002] In recent years, with the in-depth research on the location of crop quantitative trait loci, molecular marker technology has been more and more widely used in crop marker-assisted breeding by scientific research and breeding units. In crop marker-assisted breeding programs, high-throughput, rapid, and low-cost extraction of DNA from individual crop plants has always been an ideal goal for scientific researchers and breeders. In the cotton molecular marker-assisted selection work, DNA samples are generally extracted from the leaves of cotton plants. Different from the DNA of other plants, cotton leaves contain more gossypol, polysaccharides, Ning and other secondary metabolites, these metabolites are easily oxidized when the cells are broken, and irreversibly react with proteins, nuclei...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1003
Inventor 丁霄李朋波雷梦林温思钰杨六六罗晓莉秦丽霞潘转霞李换丽吴翠翠朱永红夏芝曹彩荣
Owner COTTON RES INST SHANXI ACAD OF AGRI SCI
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