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Extraction method of strawberry genome DNA

An extraction method and genome technology, applied in the field of molecular biology, can solve the problems of sample loading difficulty, low DNA yield, electrophoresis difficulty, etc., and achieve the effect of solving the difficulty of high-quality DNA extraction

Inactive Publication Date: 2015-07-29
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods are effective for very delicate tissues, but for old leaves and fruits, the final DNA samples are often very viscous, and the extracted DNA samples contain a large amount of secondary organisms that are not easily soluble in TE buffer, entangled in it The sticky matter makes it difficult to load samples and electrophoresis during DNA detection
Gel electrophoresis detection shows that the yield of DNA is very low, and the sample well or channel is very bright (see Figure 1~2 )

Method used

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  • Extraction method of strawberry genome DNA
  • Extraction method of strawberry genome DNA
  • Extraction method of strawberry genome DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] A kind of extraction method of strawberry genomic DNA, CTAB extract comprises, the CTAB of 1% (m / V), the Tris-HCl of 80mM, the EDTA of 10mM, the NaCl of 1M and the PVP of 1%; The pH value of CTAB extract is 7.5. Sterilize the CTAB extract at 110°C for 20 minutes, and add 1ml of β-mercaptoethanol to every 100ml of CTAB extract when in use. In the chloroform / isoamyl alcohol mixture used, the volume ratio of chloroform and isoamyl alcohol is 24:1.

[0029] Include the following steps,

[0030] (1) Preheat the CTAB extract to 55°C, add 5ml of the preheated CTAB extract to every 1g of strawberry fruit or leaf powder, keep it warm at 55°C for 30 minutes, shake it 3 times to mix it, and get the mixture Ⅰ ;

[0031] (2) Add 5M potassium acetate solution to the mixture I in step (1), the volume of the potassium acetate solution added is 1 / 4 of the volume of the CTAB extract in step (1), and place it in an ice bath for 1h to obtain the mixture II;

[0032] (3) Then add the c...

Embodiment 2

[0041] A kind of extraction method of strawberry genomic DNA, used CTAB extraction solution comprises, the CTAB of 3% (m / V), the Tris-HCl of 80-120mM, the EDTA of 25mM, the NaCl of 2M and the PVP of 3%; CTAB extracts The pH of the solution is 8.5. Sterilize the CTAB extract at 120°C for 40 minutes, and add 3ml of β-mercaptoethanol to every 100ml of CTAB extract when in use. In the chloroform / isoamyl alcohol mixture used, the volume ratio of chloroform and isoamyl alcohol is 24:1.

[0042] Include the following steps,

[0043] (1) Preheat the CTAB extract to 65°C, add 10ml of the preheated CTAB extract to every 1g of strawberry fruit or leaf powder, keep it at 65°C for 60 minutes, shake it 4 times to mix it, and get the mixture Ⅰ ;

[0044] (2) Add 5M potassium acetate solution to the mixture I in the step (1), the volume of the potassium acetate solution added is 1 / 3 of the volume of the CTAB extract solution in the step (1), and place it in an ice bath for 2 hours to obtai...

Embodiment 3

[0052] A kind of extraction method of strawberry genomic DNA, used CTAB extract comprises, the CTAB of 2% (m / V), the Tris-HCl of 100mM, the EDTA of 20mM, the NaCl of 1.4M and the PVP of 2%; CTAB extract The pH value is 8.0. Sterilize the CTAB extract at 121°C for 30 minutes, and add 2ml of β-mercaptoethanol to every 100ml of CTAB extract when in use. In the chloroform / isoamyl alcohol mixture used, the volume ratio of chloroform and isoamyl alcohol is 24:1.

[0053] Include the following steps,

[0054] (1) Preheat the CTAB extract to 50°C, add 8ml of the preheated CTAB extract to 1g of strawberry fruit or leaf powder, keep it warm at 60°C for 45 minutes, shake it 3 times to mix it, and get the mixture Ⅰ ;

[0055] (2) Add 5M potassium acetate solution to the mixture I in step (1), the volume of the potassium acetate solution added is 7 / 24 of the volume of the CTAB extract in step (1), place it under ice bath conditions for 1.5h, and obtain Mixture II;

[0056] (3) Then ad...

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Abstract

The invention belongs to the technical field of molecular biology, and discloses an extraction method of strawberry genome DNA. The extraction method comprises following steps: liquid nitrogen is used for smashing samples into powder; CTAB extracting solution is added; an obtained mixture is subjected to water bath at 55 to 65 DEG C and then is cooled to room temperature; potassium acetate is added, and ice bath is carried out; a chloroform / isoamyl alcohol mixed liquid of a same volume is added; an obtained mixed material is mixed uniformly, and is subjected to centrifugation; the chloroform / isoamyl alcohol mixed liquid of a same volume is added into an obtained supernate A; an obtained product is mixed uniformly, is allowed to stand, and is subjected to centrifugation so as to obtain a supernate B; isopropyl alcohol is added into the supernate B, an obtained mixed solution is subjected to centrifugation so as to obtain a precipitate; a supernate C obtained via centrifugation is removed; TE buffer containing RNase is subjected to water bath at 37 DEG C; the chloroform / isoamyl alcohol mixed liquid of a same volume is added, an obtained mixed liquid is subjected to uniform mixing, is allowed to stand, and is subjected to centrifugation so as to obtain a supernate D; sodium acetate and absolute ethyl alcohol are added into the supernate D; an obtained mixed product is subjected to centrifugation so as to remove an obtained supernate E; TE buffer is added into an obtain precipitate product so as to dissolve the precipitate product, and a strawberry genome DNA solution is obtained. According to the extraction method, characteristics of CTAB and SDS extraction methods are combined, and high quality genome DNA of strawberry which is abundant in glucose and polyphenols is obtained.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a method for extracting strawberry genome DNA. Background technique [0002] Strawberry is a perennial herb of Rosaceae, which can bloom and bear fruit many times a year, and is an important seasonal fruit in winter in my country. Strawberry leaves and fruits are rich in secondary organisms such as polysaccharides, proteins, phenolic acids, and vitamins, which make it of high utilization value, but at the same time, it also brings great difficulties to the extraction of its genomic DNA. Because these highly viscous secondary organisms will co-precipitate or bind together with DNA, which seriously affects the quality of DNA, making it difficult to carry out subsequent molecular biology such as amplification and enzyme digestion. [0003] The extraction and separation of high-quality genomic DNA is the basis of molecular biology research. The most basic extraction method...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 苗立祥张豫超蒋桂华杨肖芳肖金平王月志
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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