CN113930419A - Mango weevil DNA extraction method - Google Patents
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- CN113930419A CN113930419A CN202111209584.XA CN202111209584A CN113930419A CN 113930419 A CN113930419 A CN 113930419A CN 202111209584 A CN202111209584 A CN 202111209584A CN 113930419 A CN113930419 A CN 113930419A
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
Abstract
The invention discloses a mango weevil DNA extraction method, which comprises the following steps: step one, grinding by liquid nitrogen: selecting a mango weevil body, removing a coleoptera of the mango weevil body, and selecting a breast muscle tissue; selecting a grinding container with the matched volume according to the size of the breast muscle tissue; grinding the breast muscle tissue into powder by using liquid nitrogen in a selected grinding container to obtain a powder sample; step two, cracking digestion: adding proteinase K into a DNA lysate, and carrying out cracking digestion on the powdery sample; step three, alcohol precipitation: processing the sample subjected to cracking digestion treatment by using absolute ethyl alcohol; step four, elution: eluting the sample subjected to alcohol precipitation treatment, and collecting DNA; step five, clearing RNA: RNaseA was added to the collected DNA to eliminate RNA. The invention improves the traditional extraction method and can obtain the mango weevil DNA with high purity and high quality.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a mango weevil DNA extraction method.
Background
Mango (Mangifera indica L.) is a evergreen tree of the genus mango of the family Anacardiaceae, enjoying the reputation of "the king of tropical fruits". The mango weevil is a coleoptera weevil, and is divided into four categories of mango leaf-cutting weevil, mango pulp weevil, mango fruit weevil and mango kernel weevil, wherein the former category is mainly harmful tender leaves, and the latter three categories are listed in a two-category dangerous pest list in China, are key objects of plant quarantine, are mainly harmful fruits and pulps, and cause great loss to the mango industry.
At present, most of research on mango weevils stays in morphological observation and biological habit observation, and molecular biology research such as molecular identification and molecular resistance mechanism of mango weevils is in a primary exploration stage, so that in order to overcome uncertainty and limitation of traditional morphological research, further molecular biology research on mango weevils is urgently needed so as to rapidly and accurately identify different types of mango weevils, understand resistance mechanism of mango weevils, and then prescribe drugs for symptoms.
DNA extraction is an important link for developing the research of mango weevil molecular biology, and the classical phenol chloroform method is mostly adopted to extract insect DNA at present. The experimental result shows that when the method is used for extracting the mango weevil DNA, the problems of low extraction rate, more proteins, phenols and the like exist, the subsequent PCR amplification is influenced, and the experimental failure is easily caused, so that the rapid and efficient mango weevil DNA extraction method is urgently needed to be explored.
Disclosure of Invention
An object of the present invention is to solve at least the above problems and/or disadvantages and to provide at least the advantages described hereinafter.
The invention aims to provide a mango weevil DNA extraction method, which can improve the extraction purity and quality of mango weevil DNA.
To achieve these objects and other advantages in accordance with the present invention, there is provided a method for extracting mango weevil DNA, comprising:
step one, grinding by liquid nitrogen: selecting a mango weevil body, removing a coleoptera of the mango weevil body, and selecting a breast muscle tissue; selecting a grinding container with the matched volume according to the size of the breast muscle tissue; grinding the breast muscle tissue into powder by using liquid nitrogen in a selected grinding container to obtain a powder sample;
step two, cracking digestion: adding proteinase K into a DNA lysate, and carrying out cracking digestion on the powdery sample;
step three, alcohol precipitation: processing the sample subjected to cracking digestion treatment by using absolute ethyl alcohol;
step four, elution: eluting the sample subjected to alcohol precipitation treatment, and collecting DNA;
step five, clearing RNA: RNaseA was added to the collected DNA to eliminate RNA.
Preferably, in the method for extracting DNA of mango weevil, in the first step, the selected grinding container is a 2mL centrifuge tube or a medium-sized mortar.
Preferably, in the second step of the method for extracting DNA of mango weevil, proteinase K is added to the DNA lysate to perform lysis digestion on the powdery sample, and the specific process includes:
adding a DNA lysate A for digestion: adding 180 mu L of DNA lysate A and 20 mu L of protease K into the powdery sample, and fully and uniformly mixing; wherein the DNA lysate A is a component in the genomic DNA small extraction kit;
water bath in step (2): incubating in water bath at 55 ℃ for 1-3 h until complete cracking, and slightly shaking up once every 15 min;
adding a DNA lysate B for digestion: adding 200 mu L of DNA lysate B, fully and uniformly mixing, and carrying out water bath for 10min at the temperature of 70 ℃, wherein the DNA lysate B is a component in the genomic DNA small extraction kit.
Preferably, in the fourth step of the method for extracting DNA of mango weevil, the eluting process is performed on the sample subjected to the alcohol precipitation process, and the DNA is collected, and the specific process includes:
step (1) first elution: adding the sample subjected to alcohol precipitation into a DNA purification column, transferring all precipitates, centrifuging at 12000rmp for 1min, discarding waste liquid, adding 500 mu L of washing liquid I, centrifuging at 12000rmp for 1min, discarding waste liquid;
step (2) second elution: adding 600 μ L of washing solution II, centrifuging at 12000rmp for 1min, pouring the waste liquid, and centrifuging at 12000rmp for 1 min;
step (3) DNA collection: placing the DNA purification column on a 1.5mL centrifuge tube, adding 100 mu L of eluent, placing at room temperature for 3min, and centrifuging at 12000rmp for 1min to obtain a liquid, namely purified DNA; wherein the washing solution I, the washing solution II and the eluent are all components in the genomic DNA small-amount extraction kit.
Preferably, in the method for extracting the mango weevil DNA, in the fifth step, 2 μ L of 100mg/ml RNaseA is added to the collected DNA, mixed uniformly and placed at room temperature for 2 min.
Preferably, in the method for extracting the mango weevil DNA, in the first step, the selected mango weevil is a worm soaked in pure alcohol; and after the coleoptera of the mango weevil body is removed and the breast muscle tissue is selected, the breast muscle tissue is washed by sterile water to remove residual alcohol.
Preferably, in the method for extracting DNA of mango weevil, in the step (2), in the water bath process, the ultrasonic treatment is performed between two times of slight shaking, wherein the ultrasonic power is 80w, and the ultrasonic time is 1 s; and (3) in the digestion process of the DNA lysate B, carrying out ultrasonic treatment, wherein the ultrasonic power is 60w, the ultrasonic treatment lasts for 1s and 2min, and the circulation is finished until the water bath.
Preferably, in the first step of the method for extracting DNA of mango weevil, the breast muscle tissue is ground into powder by using liquid nitrogen in the selected grinding container to obtain a powder sample, and the specific process includes:
adding liquid nitrogen into the selected grinding container, and pre-cooling the selected grinding container and the grinding rod by utilizing the liquid nitrogen;
and (2) putting the breast muscle tissue into the selected grinding container, quickly grinding, and supplementing liquid nitrogen into the selected grinding container every 20 seconds of grinding until the breast muscle tissue is ground into powder, so as to obtain the powder sample.
The invention at least comprises the following beneficial effects:
(1) a grinding container with matched volume is selected according to the size of the worm body to grind the worm body by liquid nitrogen, so that the grinding is more precise and sufficient, and the DNA release amount is improved.
(2) Removing coleoptera of adult mango weevil, and selecting breast muscle tissue to improve grinding quality.
(3) The proteinase K is added into the DNA lysate, so that the interference of protein on DNA can be eliminated, and the extraction purity and quality are improved.
(4) The method provided by the invention improves the traditional extraction method, can obtain high-purity and high-quality mango weevil DNA, and lays a foundation for ensuring the smooth development of mango weevil molecular biology research.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
FIG. 1 is a schematic diagram of a mango weevil DNA extraction method according to an embodiment of the present invention;
FIG. 2 is a gel electrophoresis test of 2 mango weevil DNA according to example 1 of the present invention;
FIG. 3 is a gel electrophoresis test of 2 mango weevil DNA according to example 2 of the present invention.
Detailed Description
The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.
As shown in fig. 1, the present invention provides a method for extracting DNA of mango weevil, comprising:
step one, grinding by liquid nitrogen: selecting a mango weevil body, removing a coleoptera of the mango weevil body, and selecting a breast muscle tissue; selecting a grinding container with the matched volume according to the size of the breast muscle tissue; and grinding the breast muscle tissue into powder by using liquid nitrogen in a selected grinding container to obtain a powder sample.
Experiments show that the grinding has a large influence on the DNA extraction. Therefore, the grinding container with the matched volume is selected according to the size of the worm body to grind the liquid nitrogen, so that the grinding is more precise and sufficient, the DNA release amount is improved, and the DNA extraction rate is further improved. In a preferred embodiment, in the first step of the method for extracting DNA of mango weevil, the selected grinding container is a 2mL centrifuge tube or a medium-sized mortar. Preferably, the adult mango leaf cutting weevil is small in size and good in grinding quality by using a small pestle matched with a centrifugal tube, and the mango pulp weevil, the kernel weevil and the fruit weevil are good in grinding effect by using a medium-sized mortar.
In addition, when the mango weevil body is selected, the coleoptera of the mango weevil adult is removed, and the breast muscle tissue is selected, so that the grinding quality can be improved.
Step two, cracking digestion: adding proteinase K into the DNA lysate, and carrying out cracking digestion on the powdery sample.
Experiments show that the protein has a large influence on the purity of the DNA. The protein content in the mango weevil is high, and the removal of the mango weevil is difficult by conventional phenol chloroform extraction. Based on this, the embodiment of the invention utilizes proteinase K to digest protein in the mango weevil, thereby achieving the purpose of improving the extraction purity of DNA.
Step three, alcohol precipitation: and (4) processing the sample subjected to the cracking digestion treatment by using absolute ethyl alcohol.
Step four, elution: and (4) eluting the sample subjected to alcohol precipitation treatment, and collecting DNA.
Step five, clearing RNA: RNaseA was added to the collected DNA to eliminate RNA.
Here, after the step five, the method may further include the step of: and (3) detection: the extracted DNA was detected by gel electrophoresis.
In summary, the present invention mainly solves two key technical problems: selecting a proper grinding tool, discarding harder organs, and selecting proper muscle tissues, so that the problem of insufficient grinding is solved, and the release amount of DNA in the mango weevil is increased; secondly, digesting the protein in the mango weevil by using a proper amount of proteinase K, and improving the extraction purity of DNA. The invention improves the traditional extraction method, obtains the mango weevil DNA with high purity and high quality, and lays a foundation for ensuring the smooth development of the mango weevil molecular biology research.
Specifically, the invention uses a genome DNA small extraction kit (Shanghai Biyuntian biotechnology limited) to realize extraction of mango weevil DNA, and establishes a rapid and effective mango weevil DNA extraction method, wherein the operation method comprises the following steps: (1) grinding by liquid nitrogen; (2) digesting the lysate; (3) precipitating with ethanol; (4) eluting with a washing solution; (5) rnase a clears RNA; (6) detection by 1.5% agarose gel electrophoresis.
In a preferred embodiment, in the second step of the method for extracting mango weevil DNA, proteinase K is added to the DNA lysis solution to perform lysis digestion on the powdered sample, and the specific process includes:
adding a DNA lysate A for digestion: adding 180 mu L of DNA lysate A and 20 mu L of protease K into the powdery sample, and fully and uniformly mixing; wherein the DNA lysate A is a component in the genomic DNA small extraction kit;
water bath in step (2): incubating in water bath for 1-3 h at 55 ℃ until complete cracking, and slightly shaking up once every 15min in the middle to facilitate full cracking;
adding a DNA lysate B for digestion: adding 200 mu L of DNA lysate B, fully and uniformly mixing, and carrying out water bath for 10min at the temperature of 70 ℃, wherein the DNA lysate B is a component in the genomic DNA small extraction kit.
In a preferred embodiment, in the fourth step of the method for extracting DNA of mango weevil, the eluting process is performed on the sample subjected to the alcohol precipitation process, and the DNA is collected, and the specific process includes:
step (1) first elution: adding the sample subjected to alcohol precipitation into a DNA purification column, transferring all precipitates, centrifuging at 12000rmp for 1min, discarding waste liquid, adding 500 mu L of washing liquid I, centrifuging at 12000rmp for 1min, discarding waste liquid;
step (2) second elution: adding 600 μ L of washing solution II, centrifuging at 12000rmp for 1min, pouring out waste liquid, centrifuging at 12000rmp for 1min, and removing residual ethanol;
step (3) DNA collection: placing the DNA purification column on a 1.5mL centrifuge tube, adding 100 mu L of eluent, placing at room temperature for 3min, and centrifuging at 12000rmp for 1min to obtain a liquid, namely purified DNA; wherein the washing solution I, the washing solution II and the eluent are all components in the genomic DNA small-amount extraction kit.
In a preferred embodiment, in the mango weevil DNA extraction method, in the fifth step, 2 μ L of 100mg/ml RNaseA is added to the collected DNA, mixed uniformly and left at room temperature for 2 min.
In a preferred embodiment, in the method for extracting the DNA of the mango weevil, in the first step, the selected mango weevil is a worm soaked in pure alcohol; and after the coleoptera of the mango weevil body is removed and the breast muscle tissue is selected, the breast muscle tissue is washed by sterile water to remove residual alcohol.
In a preferred embodiment, in the method for extracting the mango weevil DNA, in the step (2), ultrasonic treatment is carried out in the middle of two times of slight shaking in the water bath process, wherein the ultrasonic power is 80w, and the ultrasonic time is 1 s; and (3) in the digestion process of the DNA lysate B, carrying out ultrasonic treatment, wherein the ultrasonic power is 60w, the ultrasonic treatment lasts for 1s and 2min, and the circulation is finished until the water bath. Sonication helps to promote adequate lysis, while relatively mild sonication conditions may also avoid causing DNA fragmentation. Moreover, the ultrasonic treatment conditions in the step (3) are milder than those in the water bath process in the step (2), so that the reaction is promoted while DNA fragmentation is avoided.
In a preferred embodiment, in the first step of the method for extracting DNA of mango weevil, the breast muscle tissue is ground into powder by using liquid nitrogen in a selected grinding container to obtain a powder sample, and the specific process includes:
adding liquid nitrogen into the selected grinding container, and pre-cooling the selected grinding container and the grinding rod by utilizing the liquid nitrogen;
and (2) putting the breast muscle tissue into the selected grinding container, quickly grinding, and supplementing liquid nitrogen into the selected grinding container every 20 seconds of grinding until the breast muscle tissue is ground into powder, so as to obtain the powder sample.
Wherein, under the condition of precooling grinding container and grinding rod, when the sample is put into the grinding container, it can more quickly enter into the frozen state, and then improve the quality and efficiency of grinding. In addition, in the grinding process, liquid nitrogen is supplemented to the grinding container at certain intervals to ensure that the sample is always in a frozen state and avoid DNA degradation.
Three examples are provided below to further illustrate the technical solutions provided by the present invention.
Example 1
The method for extracting the mango weevil DNA in the embodiment comprises the following steps:
(1) grinding: selecting muscle tissue with proper size from the worm body soaked in the pure alcohol, washing with sterile water, removing residual alcohol, washing completely, fully grinding the tissue into powder by using liquid nitrogen, and transposing into a 2.0mL centrifuge tube.
(2) Adding a DNA lysate for digestion: adding 180 mu LDNA lysate A and 20ul protease k, and mixing well.
(3) Water bath: incubate in 55 deg.C water bath for 1h until lysis is complete, shake gently every 15min in the middle to allow for sufficient lysis.
(4) Adding a DNA lysate for further digestion: adding 200 μ LDNA lysate B, mixing well, and water bath at 70 deg.C for 10 min.
(5) Alcohol precipitation: adding 200 μ L of absolute ethyl alcohol for precipitation, and mixing well.
(6) First elution: the mixture was applied to a DNA purification column, taking care that the precipitate had to be transferred in its entirety, centrifuged at 12000rmp for 1min, the waste solution was discarded, 500. mu.L of washing solution I was added, centrifuged at 12000rmp for 1min, and the waste solution was discarded.
(7) And (3) second elution: adding 600 μ L of washing solution II, centrifuging at 12000rmp for 1min, pouring the waste liquid, centrifuging at 12000rmp for 1min, and removing residual ethanol.
(8) Collecting DNA: placing the DNA purification column on a 1.5mL centrifuge tube, adding 100 μ L of eluent, placing at room temperature for 3min, and centrifuging at 12000rmp for 1min to obtain the liquid, namely the purified DNA.
(9) Clearing RNA: adding 2 μ L of RNaseA 100mg/ml, mixing, standing at room temperature for 2 min.
(10) And (3) detection: detecting by 1.5% agarose gel electrophoresis, and storing in a refrigerator at-20 deg.C. FIG. 2 is a gel electrophoresis detection of DNA extracted from 2 adult mango weevils. As can be seen from FIG. 2, the DNA band is single and no dispersion occurs, which indicates that this example can extract high quality and high purity DNA from adult mango weevils.
Example 2
The method for extracting the mango weevil DNA in the embodiment comprises the following steps:
(1) grinding: selecting muscle tissue with proper size from the worm body soaked in the pure alcohol, washing with sterile water, removing residual alcohol, washing completely, fully grinding the tissue into powder by using liquid nitrogen, and transposing into a 2.0mL centrifuge tube.
(2) Adding a DNA lysate for digestion: adding 180 mu LDNA lysate A and 20ul protease k, and mixing well.
(3) Water bath: the cells were incubated in a water bath at 55 ℃ for 3h to complete lysis and gently shaken every 15min in the middle to allow for sufficient lysis.
(4) Adding a DNA lysate for further digestion: adding 200 μ LDNA lysate B, mixing well, and water bath at 70 deg.C for 10 min.
(5) Alcohol precipitation: adding 200 μ L of absolute ethyl alcohol for precipitation, and mixing well.
(6) First elution: the mixture was applied to a DNA purification column, taking care that the precipitate had to be transferred in its entirety, centrifuged at 12000rmp for 1min, the waste solution was discarded, 500. mu.L of washing solution I was added, centrifuged at 12000rmp for 1min, and the waste solution was discarded.
(7) And (3) second elution: adding 600 μ L of washing solution II, centrifuging at 12000rmp for 1min, pouring the waste liquid, centrifuging at 12000rmp for 1min, and removing residual ethanol.
(8) Collecting DNA: placing the DNA purification column on a 1.5mL centrifuge tube, adding 100 μ L of eluent, placing at room temperature for 3min, and centrifuging at 12000rmp for 1min to obtain the liquid, namely the purified DNA.
(9) Clearing RNA: adding 2 μ L of RNaseA 100mg/ml, mixing, standing at room temperature for 2 min.
(10) And (3) detection: detecting by 1.5% agarose gel electrophoresis, and storing in a refrigerator at-20 deg.C. FIG. 3 is a gel electrophoresis detection of DNA extracted from 2 adult mango weevils. As can be seen from FIG. 3, the DNA band is single and no dispersion occurs, indicating that this example can extract high quality and high purity DNA from adult mango weevils.
Example 3
The method for extracting the mango weevil DNA in the embodiment comprises the following steps:
(1) grinding: selecting muscle tissue with proper size from the worm body soaked in pure alcohol, and washing with sterile water to remove residual alcohol. After the breast muscle tissue is washed clean, liquid nitrogen is added into the grinding container, the selected grinding container and the selected grinding rod are pre-cooled by the liquid nitrogen, the breast muscle tissue is placed into the selected grinding container, the breast muscle tissue is quickly ground, the liquid nitrogen is supplemented into the selected grinding container every 20 seconds of grinding until the breast muscle tissue is ground into powder, a powder sample is obtained, and the powder sample is transferred to a 2.0mL centrifuge tube.
(2) Adding a DNA lysate for digestion: adding 180 mu LDNA lysate A and 20ul protease k, and mixing well.
(3) Water bath: incubating in 55 ℃ water bath for 1h until complete lysis, slightly shaking up every 15min, and carrying out ultrasonic treatment in the middle of slightly shaking up twice, wherein the ultrasonic power is 80w, and the ultrasonic is 1 s.
(4) Adding a DNA lysate for further digestion: adding 200 μ LDNA lysate B, mixing, performing water bath at 70 deg.C for 10min, performing ultrasonic treatment with ultrasonic power of 60w for 1s, stopping for 2min, and circulating until the water bath is finished.
(5) Alcohol precipitation: adding 200 μ L of absolute ethyl alcohol for precipitation, and mixing well.
(6) First elution: the mixture was applied to a DNA purification column, taking care that the precipitate had to be transferred in its entirety, centrifuged at 12000rmp for 1min, the waste solution was discarded, 500. mu.L of washing solution I was added, centrifuged at 12000rmp for 1min, and the waste solution was discarded.
(7) And (3) second elution: adding 600 μ L of washing solution II, centrifuging at 12000rmp for 1min, pouring the waste liquid, centrifuging at 12000rmp for 1min, and removing residual ethanol.
(8) Collecting DNA: placing the DNA purification column on a 1.5mL centrifuge tube, adding 100 μ L of eluent, placing at room temperature for 3min, and centrifuging at 12000rmp for 1min to obtain the liquid, namely the purified DNA.
(9) Clearing RNA: adding 2 μ L of RNaseA 100mg/ml, mixing, standing at room temperature for 2 min.
(10) And (3) detection: detecting by 1.5% agarose gel electrophoresis, and storing in a refrigerator at-20 deg.C. The detection shows that the DNA strip is single and no dispersion occurs, which indicates that the embodiment can extract high-quality and high-purity DNA from adult mango weevils.
While embodiments of the invention have been disclosed above, it is not intended to be limited to the uses set forth in the specification and examples. It can be applied to all kinds of fields suitable for the present invention. Additional modifications will readily occur to those skilled in the art. It is therefore intended that the invention not be limited to the exact details and illustrations described and illustrated herein, but fall within the scope of the appended claims and equivalents thereof.
Claims (8)
1. A mango weevil DNA extraction method is characterized by comprising the following steps:
step one, grinding by liquid nitrogen: selecting a mango weevil body, removing a coleoptera of the mango weevil body, and selecting a breast muscle tissue; selecting a grinding container with the matched volume according to the size of the breast muscle tissue; grinding the breast muscle tissue into powder by using liquid nitrogen in a selected grinding container to obtain a powder sample;
step two, cracking digestion: adding proteinase K into a DNA lysate, and carrying out cracking digestion on the powdery sample;
step three, alcohol precipitation: processing the sample subjected to cracking digestion treatment by using absolute ethyl alcohol;
step four, elution: eluting the sample subjected to alcohol precipitation treatment, and collecting DNA;
step five, clearing RNA: RNaseA was added to the collected DNA to eliminate RNA.
2. The method for extracting mango weevil DNA as claimed in claim 1, wherein in the first step, the selected grinding container is a 2mL centrifuge tube or a medium-sized mortar.
3. The method for extracting mango weevil DNA as set forth in claim 1, wherein in the second step, proteinase K is added to the DNA lysis solution to perform lysis digestion on the powdery sample, and the specific process comprises:
adding a DNA lysate A for digestion: adding 180 mu L of DNA lysate A and 20 mu L of protease K into the powdery sample, and fully and uniformly mixing; wherein the DNA lysate A is a component in the genomic DNA small extraction kit;
water bath in step (2): incubating in water bath at 55 ℃ for 1-3 h until complete cracking, and slightly shaking up once every 15 min;
adding a DNA lysate B for digestion: adding 200 mu L of DNA lysate B, fully and uniformly mixing, and carrying out water bath for 10min at the temperature of 70 ℃, wherein the DNA lysate B is a component in the genomic DNA small extraction kit.
4. The method for extracting mango weevil DNA as claimed in claim 1, wherein in the fourth step, the sample subjected to alcohol precipitation is eluted and DNA is collected, and the specific process comprises the following steps:
step (1) first elution: adding the sample subjected to alcohol precipitation into a DNA purification column, transferring all precipitates, centrifuging at 12000rmp for 1min, discarding waste liquid, adding 500 mu L of washing liquid I, centrifuging at 12000rmp for 1min, discarding waste liquid;
step (2) second elution: adding 600 μ L of washing solution II, centrifuging at 12000rmp for 1min, pouring the waste liquid, and centrifuging at 12000rmp for 1 min;
step (3) DNA collection: placing the DNA purification column on a 1.5mL centrifuge tube, adding 100 mu L of eluent, placing at room temperature for 3min, and centrifuging at 12000rmp for 1min to obtain a liquid, namely purified DNA; wherein the washing solution I, the washing solution II and the eluent are all components in the genomic DNA small-amount extraction kit.
5. The method for extracting mango weevil DNA as set forth in claim 1, wherein in the fifth step, 2. mu.L of 100mg/ml RNaseA is added to the collected DNA, mixed, and left at room temperature for 2 min.
6. The mango weevil DNA extraction method according to claim 1, wherein in the first step, the selected mango weevils are worms soaked in pure alcohol; and after the coleoptera of the mango weevil body is removed and the breast muscle tissue is selected, the breast muscle tissue is washed by sterile water to remove residual alcohol.
7. The mango weevil DNA extraction method according to claim 3, wherein in the step (2), the ultrasonic treatment is carried out in the middle of two times of slight shaking in the water bath process, wherein the ultrasonic power is 80w and the ultrasonic time is 1 s; and (3) in the digestion process of the DNA lysate B, carrying out ultrasonic treatment, wherein the ultrasonic power is 60w, the ultrasonic treatment lasts for 1s and 2min, and the circulation is finished until the water bath.
8. The method for extracting mango weevil DNA as claimed in claim 1, wherein in the first step, the breast muscle tissue is ground into powder by liquid nitrogen in a selected grinding container to obtain a powder sample, and the specific process comprises the following steps:
adding liquid nitrogen into the selected grinding container, and pre-cooling the selected grinding container and the grinding rod by utilizing the liquid nitrogen;
and (2) putting the breast muscle tissue into the selected grinding container, quickly grinding, and supplementing liquid nitrogen into the selected grinding container every 20 seconds of grinding until the breast muscle tissue is ground into powder, so as to obtain the powder sample.
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