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Method for effectively extracting genomic DNA (deoxyribonucleic acid) of melaleuca alternifolia

A Melaleuca alternifolia and genome technology, applied in the field of plant DNA extraction, can solve the problems of low DNA yield, poor DNA quality, and high cost, and achieve the effects of enhancing safety, saving costs, and saving experimental operation time

Inactive Publication Date: 2018-08-17
GUANGXI FORESTRY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] At present, there is no report on the method of extracting Melaleuca alternifolia genomic DNA. When the conventional SDS method and CTAB method are used to extract Melaleuca alternifolia genomic DNA, the quality of the obtained DNA is poor and the effect of PCR amplification is poor. Poor, and the use of kits to extract Melaleuca alternifolia genomic DNA, the cost is high and the DNA yield is low

Method used

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  • Method for effectively extracting genomic DNA (deoxyribonucleic acid) of melaleuca alternifolia
  • Method for effectively extracting genomic DNA (deoxyribonucleic acid) of melaleuca alternifolia
  • Method for effectively extracting genomic DNA (deoxyribonucleic acid) of melaleuca alternifolia

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Effect test

Embodiment 1

[0034] A method for effectively extracting Melaleuca alternifolia genomic DNA, specifically comprising the steps of:

[0035] (1) Grind 1 g fresh leaf sample of Melaleuca alternifolia with liquid nitrogen to powder, quickly transfer to a centrifuge tube, add 600 µl of conventional preheated DNA extraction buffer, mix well, and place in a water bath at 65°C for 30 minutes, in which the DNA extraction buffer The formula of the solution is: 2% CTAB, 100mM Tris-HCl with a pH of 8.0, 20mM EDTA with a pH of 8.0, and 1M NaCl;

[0036] (2) Take out the centrifuge tube and cool to room temperature, add a mixture of chloroform and isoamyl alcohol equal to the volume of the DNA extraction buffer, the volume ratio of the mixture is: chloroform:isoamyl alcohol=24:1, mix well and place at 12000 r / min Centrifuge for 10min;

[0037] (3) Take the supernatant and put it in a new centrifuge tube, add the same volume of chloroform as the supernatant, mix well and centrifuge at 12000 r / min for 1...

Embodiment 2

[0044] A method for effectively extracting Melaleuca alternifolia genomic DNA, specifically comprising the steps of:

[0045] (1) Grind 2 g of fresh leaves of Melaleuca alternifolia to powder with liquid nitrogen, quickly transfer to a centrifuge tube, add 800 µl of conventional preheated DNA extraction buffer, mix well, and place in a water bath at 65°C for 30 minutes; the DNA extraction buffer The formula of the solution is: 2% CTAB, 100mM Tris-HCl with a pH of 8.0, 20mM EDTA with a pH of 8.0, and 1M NaCl;

[0046] (2) Take out the centrifuge tube and cool to room temperature, add a mixture of chloroform and isoamyl alcohol equal to the volume of the DNA extraction buffer, the volume ratio of the mixture is: chloroform:isoamyl alcohol=24:1, mix well and place at 12000 r / min Centrifuge for 10min;

[0047] (3) Take the supernatant and put it in a new centrifuge tube, add the same volume of chloroform as the supernatant, mix well and centrifuge at 12000 r / min for 10 minutes;

...

Embodiment 3

[0054] A method for effectively extracting Melaleuca alternifolia genomic DNA, specifically comprising the steps of:

[0055] (1) Grind 1.5g fresh leaf samples of Melaleuca alternifolia to powder form with liquid nitrogen, quickly transfer to a centrifuge tube, add 700µl conventional preheated DNA extraction buffer, mix well, and place in a water bath at 65°C for 30 minutes; DNA extraction The formula of the buffer solution is: 2% CTAB, 100mM Tris-HCl with a pH of 8.0, 20mM EDTA with a pH of 8.0, and 1M NaCl;

[0056] (2) Take out the centrifuge tube and cool to room temperature, add a mixture of chloroform and isoamyl alcohol equal to the volume of the DNA extraction buffer, the volume ratio of the mixture is: chloroform:isoamyl alcohol=24:1, mix well and place at 12000 r / min Centrifuge for 10min;

[0057] (3) Take the supernatant and put it in a new centrifuge tube, add the same volume of chloroform as the supernatant, mix well and centrifuge at 12000 r / min for 10 minutes;...

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Abstract

The invention provides a method for effectively extracting genomic DNA (deoxyribonucleic acid) of melaleuca alternifolia, which comprises the following steps: (1) grinding a sample into powder by liquid nitrogen, transferring the powder to a centrifuge tube, adding a DNA extraction buffer which is conventionally preheated, uniformly mixing and carrying out water bath at 65 DEG C for 30 minutes; (2) taking out the centrifuge tube and cooling to room temperature, adding a mixed solution of chloroform and isoamyl alcohol, uniformly mixing and performing centrifugal separation; (3) taking supernate, adding chloroform, uniformly mixing and performing centrifugal separation; (4) taking supernate, adding absolute ethyl alcohol which is conventionally precooled, uniformly mixing, standing for 30 minutes at -20 DEG C, then performing centrifugal separation, and discarding the supernate; (5) washing sedimentation with ethyl alcohol of which the volume concentration is 75%, drying at room temperature and adding a DNA dissolution buffer, and storing at -20 DEG C for standby use. The method provided by the invention is capable of effectively and rapidly extracting the genomic DNA of the melaleuca alternifolia which is high in purity and suitable for PCR detection, and the use of toxic chemicals in the extracting process is reduced.

Description

technical field [0001] The invention belongs to the technical field of plant DNA extraction, and in particular relates to a genome DNA extraction method of Melaleuca alternifolia. Background technique [0002] Melaleuca alternifolia ( Melaleuca alternifolia ) is the myrtaceae ( Myrlaceaca ) Melaleuca (Melaleuca) evergreen shrub to small tree species, naturally distributed in Queensland and northern New South Wales in northeastern Australia. Essential oil can be extracted from the fresh branches and leaves of Melaleuca alternifolia, which has good bactericidal effect and is widely used in food, pharmaceutical, spice and other industries. [0003] Melaleuca alternifolia essential oil is divided into three different biochemical types according to different components. According to different utilization purposes, choosing to introduce different biochemical types of Melaleuca alternifolia can avoid unnecessary economic losses. However, it is difficult to distinguish the phen...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/6806
CPCC12N15/1003C12Q1/6806
Inventor 邓紫宇唐庆兰陈健波刘媛卢翠香郭东强李昌荣任世奇项东云周维伍琪梁忠云
Owner GUANGXI FORESTRY RES INST
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