CN106086005A

CN106086005A - A kind of Wheat DNA rapid extracting method

Info

Publication number: CN106086005A
Application number: CN201610703947.8A
Authority: CN
Inventors: 朱永兴; 关雅静; 郭生虎; 石磊; 白海波; 张岩
Original assignee: Ningxia Academy of Agriculture and Forestry Sciences
Current assignee: Ningxia Academy of Agriculture and Forestry Sciences
Priority date: 2016-08-22
Filing date: 2016-08-22
Publication date: 2016-11-09

Abstract

This application discloses a kind of Wheat DNA rapid extracting method, comprise the following steps: the configuration of extracting solution；Weigh wheat leaf blade tissue, be milled to powder with liquid nitrogen, go in centrifuge tube stand-by；Add extracting solution in centrifuge tube, mixing, then add saturated phenol, continue mixing, continuously add chloroform, continue high speed centrifugation after mixing；Aspirate supernatant continuously adds chloroform to new centrifuge tube, continues high speed centrifugation after mixing；Aspirate supernatant adds high speed centrifugation after aqueous isopropanol mixes to new centrifuge tube, abandons supernatant, by precipitate with alcohol flushing once, dries, and adds ultra-pure water and dissolves.The application operating procedure is simple, shortens the time of half than prior art；It is not easy to degraded, beneficially the carrying out of subsequent experimental.

Description

A kind of Wheat DNA rapid extracting method

Technical field

The application belongs to biological heredity field, specifically, relates to a kind of Wheat DNA rapid extracting method.

Background technology

It is operation most important, most basic in Marker-assisted selection that DNA of plants is extracted. in order to isolate high-quality, high-purity The Plant Genome of degree

DNA, many scholars with the methods such as SDs (sodium lauryl sulphate), cTAB (cetyltriethylammonium bromide) with Semen Tritici aestivi, Oryza sativa L., the tender leaf of Soybean and Other Crops are material extraction DNA, although the DNA mass separated, purity are the highest, meet PcR The requirement of detection, but this method liquid nitrogen freezing to be used, operating procedure is complicated, have impact on the universal of molecular marking technique and promotes. or not DNA_l directly can also be extracted from Semen sojae atricolor, Semen Maydis, wheat seed with liquid nitrogen]. carry from Semen Tritici aestivi, cotton leaf without liquid nitrogen Take DNA also to have been reported that；After Molecular Marker Assisted Selection Technology is universal, people need the side of simpler, easy extraction DNA Method. this research combines the advantage of several DNA extraction method on the basis of traditional plant genome DNA extracting method, explores The method that is material DNA rapid extraction with Semen Tritici aestivi spire, it is intended to for PCR amplification and the molecular marker of Main Agronomic Characters gene Assisted selection provides feasible method.

The current existing Wheat volatiles DNA extraction method time is long, and operating procedure is many, degradable, and purity is the most high to be lacked Point, present invention sets forth the operational approach solving problem above, it is intended to set up a set of easy operation, and the time is short, it is high to extract quality Wheat DNA extracting method.

Summary of the invention

In view of this, the application is long for extraction time present in prior art, serious problem of degrading, it is provided that Plant Wheat DNA rapid extracting method.

In order to solve above-mentioned technical problem, this application discloses a kind of Wheat DNA rapid extracting method, including following step Rapid:

1) configuration of extracting solution；

2) weigh 0.5-1g wheat leaf blade tissue, be milled to powder with liquid nitrogen, go in 2-5ml centrifuge tube stand-by；

3) 0.5-1ml extracting solution is added in centrifuge tube, mixing, then add the saturated phenol of 0.5-1.5ml, continue mixed Even, continuously add 0.4-1.6ml chloroform, continue high speed centrifugation after mixing；

4) Aspirate supernatant continuously adds 0.4-1.6ml chloroform to new centrifuge tube, continues high speed centrifugation after mixing；

5) Aspirate supernatant is to adding high speed centrifugation after the aqueous isopropanol mixing of 0.4-1.5ml in new centrifuge tube, abandons Supernatant, by precipitate with 100-300ml alcohol flushing once, dries, and adds 30-60uL ultra-pure water and dissolves.

Further, step 1) in the configuration of extracting solution specific as follows: by 1-10ml 1-5MLiCl solution, 1-10% SDS 10-50ml, 0.1-3M Tris.Cl 1-60ml, 0.1-2EDTA 1-15ml, be settled to 100-300ml with 50-70 DEG C, Add 100-400ml RNA digestive enzyme.

Further, step 3) in centrifugation time be 5-15min.

Further, step 4) in centrifugation time be 5-15min.

Further, step 5) in centrifugation time be 5-15min.

Further, step 5) in the mass concentration of ethanol be 70-75%.

Compared with prior art, the application can obtain and include techniques below effect:

1) operating procedure is simple, shortens the time of half than prior art.

2) it is not easy to degraded, beneficially the carrying out of subsequent experimental.

3) the Wheat DNA electrophoretic band that the present invention extracts is complete, and non-degradable, impurity is few, purity is high, and (experiment impurity is few, RNA Pollute low).

Certainly, the arbitrary product implementing the application must be not necessarily required to reach all the above technique effect simultaneously.

Accompanying drawing explanation

Fig. 1 is the electrophoretogram of the Wheat DNA using the present invention to extract, and wherein, 1-6 represents the Semen Tritici aestivi utilizing the present invention to extract DNA；

Fig. 2 is the electrophoretogram of the Wheat DNA using the most conventional method to extract, and wherein, wherein, 1-5 represents with at present The Wheat DNA that conventional method is extracted.

Detailed description of the invention

Describe presently filed embodiment in detail below in conjunction with drawings and Examples, thereby how the application is applied Technological means solves technical problem and reaches the process that realizes of technology effect and can fully understand and implement according to this.

Embodiment 1 Wheat DNA rapid extracting method

1) configuration of extracting solution: by 1-10ml 1-5MLiCl solution, 1-10%SDS 10-50ml, 0.1-3M Tris.Cl 1-60ml, 0.1-2EDTA 1-15ml, is settled to 100-300ml with 50-70 DEG C, adds 100-400ml RNA digestive enzyme；

3) 0.5-1ml extracting solution is added in centrifuge tube, mixing, then add the saturated phenol of 0.5-1.5ml, continue mixed Even, continuously add 0.4-1.6ml chloroform, continue high speed centrifugation 5-15min after mixing；

4) Aspirate supernatant continuously adds 0.4-1.6ml chloroform to new centrifuge tube, continues high speed centrifugation 5-after mixing 15min；

5) Aspirate supernatant is to adding high speed centrifugation 5-after the aqueous isopropanol mixing of 0.4-1.5ml in new centrifuge tube 15min, abandons supernatant, by precipitate with 100-300ml 70-75% alcohol flushing once, dries, and adds 30-60uL ultra-pure water Dissolve.

From Fig. 1 and Fig. 2, the Wheat DNA electrophoretic band extracted by the present invention is complete, not degraded, and does not exist miscellaneous Band pollutes, and the Wheat DNA electrophoresis that conventional method is extracted has a large amount of disperse, there is degraded, and miscellaneous band is more, is unfavorable for follow-up test Carrying out.Therefore, the Wheat DNA electrophoretic band that the present invention extracts is complete, and non-degradable, impurity is few, purity is high, and extraction time ratio Conventional method reduces half, has feature efficient, high-quality, can be used for regular-PCR detection, Southern bolt, genome The tests such as order-checking.

Described above illustrate and describes some preferred embodiments of invention, but as previously mentioned, it should be understood that invention is not It is confined to form disclosed herein, is not to be taken as the eliminating to other embodiments, and can be used for other combinations various, amendment And environment, and can be carried out by above-mentioned teaching or the technology of association area or knowledge in invention contemplated scope described herein Change.And the change that those skilled in the art are carried out and change are without departing from the spirit and scope of invention, the most all should weigh appended by invention In the protection domain that profit requires.

Claims

1. a Wheat DNA rapid extracting method, it is characterised in that comprise the following steps:

1) configuration of extracting solution；

3) 0.5-1ml extracting solution is added in centrifuge tube, mixing, then add the saturated phenol of 0.5-1.5ml, continue mixing, continue The continuous 0.4-1.6ml chloroform that adds, high speed centrifugation after continuation mixing；

5) Aspirate supernatant is to adding high speed centrifugation after the aqueous isopropanol mixing of 0.4-1.5ml in new centrifuge tube, abandons supernatant Liquid, by precipitate with 100-300ml alcohol flushing once, dries, and adds 30-60uL ultra-pure water and dissolves.

Wheat DNA rapid extracting method the most according to claim 1, it is characterised in that described step 1) in extracting solution Configuration specific as follows: by 1-10ml 1-5MLiCl solution, 1-10%SDS10-50ml, 0.1-3M Tris.Cl 1-60ml, 0.1-2EDTA 1-15ml, is settled to 100-300ml with 50-70 DEG C, adds 100-400ml RNA digestive enzyme.

Wheat DNA rapid extracting method the most according to claim 1, it is characterised in that described step 3) in centrifugal time Between be 5-15min.

Wheat DNA rapid extracting method the most according to claim 1, it is characterised in that described step 4) in centrifugal time Between be 5-15min.

Wheat DNA rapid extracting method the most according to claim 1, it is characterised in that described step 5) in centrifugal time Between be 5-15min.

Wheat DNA rapid extracting method the most according to claim 1, it is characterised in that described step 5) in ethanol Mass concentration is 70-75%.