CN116064501A - Crab meat DNA extraction kit and extraction method - Google Patents

Abstract

The invention discloses a crab meat DNA extraction kit and an extraction method, and belongs to the field of molecular biology. The kit comprises a lysate, wherein the lysate comprises SDS, proteinase K and RNase A. The kit has the advantages of simple components, easy preparation, low cost and no toxicity. The extraction method is simple and convenient, is easy to operate, can rapidly extract the DNA of the crab meat, and further can promote the high-quality and sustainable development of the aquaculture industry.

Description

Crab meat DNA extraction kit and extraction method

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to a crab meat DNA extraction kit and an extraction method.

Background

China is used as a large country for aquaculture, the total yield of aquatic products accounts for one third of the world, and the yield of aquaculture accounts for about 60% of the world's yield of aquaculture. The rapid development of aquaculture is not supported by biotechnology. The molecular biology method researches the structure and the function of the biological gene by tracing the nucleotide sequence of the biological gene, and is helpful for exploring the breeding and the reproduction of aquatic products, diseases, immunity and the like. Especially, people favor aquatic foods, so that more and more research hotspots focus on breeding and cultivation of aquatic organisms. At present, aquaculture has problems such as few genetically improved varieties, serious diseases, low breeding rate and the like.

The qualified sample DNA should have the integrity of the primary structure of the nucleic acid and reduce contamination of RNA, proteins, polysaccharides, etc. Aquatic organisms such as crabs have loose and fragile muscles and contain high proteins. Because of the characteristics of cells and tissues different from those of land organisms, the conventional DNA extraction method cannot obtain high-quality crab meat DNA.

Disclosure of Invention

In order to solve at least one of the technical problems, the invention adopts the following technical scheme:

the invention provides a crab meat DNA extraction kit, which comprises a lysate, wherein the lysate comprises SDS, proteinase K and RNase A.

In some embodiments of the invention, the lysate comprises 0.6-2.4% SDS, 2% proteinase K and 1% RNase A.

In some preferred embodiments of the invention, the lysate comprises 1% SDS, 2% proteinase K and 1% RNase A.

In some embodiments of the invention, the kit further comprises a rinse solution and an eluent.

In some embodiments of the invention, the rinse solution is an organic alcohol solution. In some preferred embodiments of the invention, the rinse solution is 80% ethanol.

In some embodiments of the invention, the eluent is selected from one of TE buffer, tris-HCl buffer and water. In some preferred embodiments of the invention, the eluent is 10mM Tris-HCl.

In some embodiments of the invention, the kit further comprises proteinase K.

The second aspect of the invention provides a method for extracting crab meat DNA by using a crab meat DNA extraction kit, which comprises the following steps:

s1, adding the cracking liquid according to the first aspect of the invention into a grinding pipe containing steel balls, taking a proper amount of crab meat into the grinding pipe, and grinding for 1-3 min by using a grinding instrument at 50 Hz; the method comprises the steps of carrying out a first treatment on the surface of the

S2, placing the grinding sample tube in a water bath kettle at 65 ℃ for 13-15 min;

s3, placing the grinding sample tube in a water bath kettle at 70 ℃ for 13-15 min;

s4, placing the grinding sample tube into a centrifugal machine 8000-10000 g to be centrifuged for 10-15 min, and transferring the supernatant into a new centrifugal tube;

s5, adding chloroform with the volume of 0.5 times, reversing and uniformly mixing until the solution is completely emulsified into white, and centrifuging for 8-15 min at the temperature of 12000-15000 Xg and the temperature of 4 ℃;

s6, sucking supernatant fluid and transferring the supernatant fluid into a new sterilizing centrifuge tube;

s7, adding magnetic beads with the volume of 0.6 times of that of the supernatant fluid, uniformly mixing by blowing, and standing at room temperature for 3-8 min;

s8, placing the centrifuge tube in a magnetic rack until the solution is clear, and sucking and discarding the supernatant;

s9, keeping the centrifuge tube on a magnetic rack, adding 200 mu L of rinsing liquid, standing at room temperature for 20-40S, and then sucking and discarding the supernatant;

s10, performing instantaneous centrifugation, discarding redundant rinsing liquid in the centrifuge tube, and drying the magnetic beads at room temperature for 2-5 min;

s11, taking down the centrifuge tube from the magnetic rack, and standing at room temperature for 2-5 min; adding 100 mu L of eluent, lightly blowing and mixing by a pipettor, and standing for 3-8 min at room temperature;

s12, placing the centrifuge tube on a magnetic rack, standing at room temperature for 3-8 min until the solution is clear, sucking the supernatant, and transferring the supernatant into a corresponding new sample storage tube to obtain purified crab meat DNA.

In some embodiments of the invention, step S9 is repeated one time before step S10.

In some embodiments of the invention, the rinse solution is 80% ethanol.

In some embodiments of the invention, the eluent is selected from one of TE buffer, tris-HCl buffer and water.

The beneficial effects of the invention are that

Compared with the prior art, the invention has the following beneficial effects:

the kit has the advantages of simple components, easy preparation, low cost and no toxicity.

The extraction method is simple and convenient, is easy to operate, and can rapidly extract the DNA of the crab meat.

The invention provides a high-efficiency high-quality extraction method for extracting the crab meat DNA, and can promote the high-quality and sustainable development of the aquaculture industry.

Drawings

FIG. 1 shows the agarose gel detection results of crab meat DNA extracted using lysate 1 formulated in example 1 in combination with the extraction method of example 2. M: DNA Marker, 1-3: sample number.

FIG. 2 shows the agarose gel detection results of crab meat DNA extracted using lysate 2 formulated in example 1 in combination with the extraction method of example 2. M: DNA Marker, 1-3: sample number.

FIG. 3 shows the agarose gel detection results of the crab meat DNA extracted according to the method provided by the kit, from the "blood/cell/tissue genomic DNA extraction kit (DP 304)" of the root. M: DNA Marker, 1-3: sample number.

FIG. 4 shows agarose gel detection results of crab meat DNA extracted using lysates containing different concentrations of SDS in combination with the extraction method of example 2. . M: DNA Marker,1: SDS concentration was 0.2%;2: SDS concentration was 0.6%;3: SDS concentration was 2.4%;4: SDS concentration was 3.2%.

Detailed Description

Unless otherwise indicated, implied from the context, or common denominator in the art, all parts and percentages in the present application are based on weight and the test and characterization methods used are synchronized with the filing date of the present application. Where applicable, the disclosure of any patent, patent application, or publication referred to in this application is incorporated by reference in its entirety, and the equivalent patents to those cited are incorporated by reference, particularly as they relate to the definitions of terms in the art. If the definition of a particular term disclosed in the prior art does not conform to any definition provided in this application, the definition of that term provided in this application controls.

Numerical ranges in this application are approximations, so that it may include the numerical values outside of the range unless otherwise indicated. The numerical range includes all values from the lower value to the upper value that increase by 1 unit, provided that there is a spacing of at least 2 units between any lower value and any higher value. For ranges containing values less than 1 or containing fractions greater than 1 (e.g., 1.1,1.5, etc.), then 1 unit is suitably considered to be 0.0001,0.001,0.01, or 0.1. For a range containing units of less than 10 (e.g., 1 to 5), 1 unit is generally considered to be 0.1. These are merely specific examples of what is intended to be provided, and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application.

The terms "comprises," "comprising," "including," and their derivatives do not exclude the presence of any other component, step or procedure, and are not related to whether or not such other component, step or procedure is disclosed in the present application. For the avoidance of any doubt, all use of the terms "comprising," "including," or "having" herein, unless expressly stated otherwise, may include any additional additive, adjuvant, or compound. Rather, the term "consisting essentially of … …" excludes any other component, step or process from the scope of any of the terms recited below, as those out of necessity for operability. The term "consisting of … …" does not include any components, steps or processes not specifically described or listed. The term "or" refers to the listed individual members or any combination thereof unless explicitly stated otherwise.

In order to make the technical problems, technical schemes and beneficial effects solved by the invention more clear, the invention is further described in detail below with reference to the embodiments.

Examples

The following examples are presented herein to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit or scope of the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, the disclosure of which is incorporated herein by reference as is commonly understood by reference.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

The experimental methods in the following examples are conventional methods unless otherwise specified. The instruments used in the following examples are laboratory conventional instruments unless otherwise specified; the test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.

Example 1 crab meat DNA extraction reagent

The extraction reagent comprises a lysate, a rinse solution and an eluent, and is respectively as follows:

1. lysate solution

In this example, 2 lysates were prepared in order to obtain the best extraction results.

Lysate 1:1mL of 2.4% SDS, 20. Mu.L of proteinase K, 10. Mu.L of RNase A;

lysate 2:1mL of 2.4% CTAB, 20. Mu.L of proteinase K, 10. Mu.L of RNase A.

2. Rinsing liquid

80% ethanol.

3. Eluent (eluent)

10mM Tris-HCl。

Example 2 crab meat DNA extraction method

Firstly, preparing 2mL of grinding sample pipes, and sequentially adding sterilized steel balls with diameters of 6 mm/3 mm respectively.

(1) Adding 1mL of a lysate (the lysate 1 or the lysate 2 prepared in the example 1) into a grinding pipe, taking a certain amount (about 500 mg) of crab meat of Eriocheir sinensis into the grinding pipe by scissors and tweezers, and grinding for 3min by using a grinder at 50 Hz;

(2) Placing the grinding sample tube into a water bath kettle at 65 ℃ for water bath for 15min;

(3) Placing the grinding sample tube into a water bath kettle at 70 ℃ for water bath for 15min;

(4) Placing the grinding sample tube into a centrifugal machine 8000g for centrifugation for 15min, and transferring the supernatant into a new 2mL centrifugal tube;

(5) Adding chloroform with the volume about 0.5 times of the supernatant into the centrifuge tube, and reversing and uniformly mixing or uniformly mixing by vortex until the solution is completely emulsified into white;

(6) Putting the centrifuge tube into a centrifuge, centrifuging 13000g for 10min at 4 ℃, and transferring the supernatant into a new 1.5mL centrifuge tube;

(7) Adding magnetic beads (Hangzhou Liangchuan biotechnology Co., ltd.) with the volume of supernatant of about 0.6 times into the centrifuge tube, blowing and mixing uniformly, and standing at room temperature for 5min;

(8) Placing the centrifuge tube on a magnetic rack for standing for 5min until the solution is clear, and discarding the supernatant;

(9) Keeping the centrifuge tube on a magnetic rack, adding 200 mu L of rinsing liquid, standing at room temperature for 30s, and removing the supernatant;

(10) Repeating the steps for one time;

(11) Taking down the centrifuge tube from the magnetic rack, and drying the magnetic beads at room temperature for 5min;

(12) Adding 100 mu L of eluent, blowing and mixing uniformly, suspending magnetic beads, and standing for 5min at room temperature;

(13) Placing the centrifuge tube on a magnetic rack for standing for 5min until the solution is clear, sucking the supernatant, transferring to a corresponding new sample preservation tube, and preserving at-20deg.C.

The DNA of 3 crab meat samples was extracted by using lysate 1 and lysate 2, and the agarose gel detection results are shown in FIG. 1 and FIG. 2, respectively. Meanwhile, the inventors also used the Tiangen "blood/cell/tissue genomic DNA extraction kit (DP 304)", DNA extraction of 3 crab meat samples according to the method provided by the kit, and agarose gel detection results are shown in FIG. 3.

From this, it was found that the maximum amount of DNA sample was obtained by extracting crab meat DNA using the method of this example using lysate 1.

The inventors further measured the concentration and OD260/OD280 of each DNA sample. The measurement results are shown in Table 1:

TABLE 1 concentration and OD260/OD280 of each DNA sample

As is clear from Table 1, the concentration of the DNA sample obtained by the extraction of the lysate 1 prepared in example 1 was highest, which could reach 212.2 ng/. Mu.L, and the purity was highest, by combining the extraction methods of this example.

Example 3 Effect of lysate formulation on crab meat DNA extraction

In order to further improve the extraction effect of crab meat DNA, the inventor further optimizes the formula of the lysate, specifically adjusts the concentration of SDS, increases proteinase K and the like. The detailed scheme is as follows:

the crab meat DNA extraction was performed according to the above method by preparing lysis solutions containing 0.2%, 0.6%, 2.4% and 3.2% SDS concentrations, respectively. The final DNA samples were assayed for sample concentration and OD260/OD280 and agarose gel detection was performed.

The results are shown in figure 4 and table 2,

TABLE 2 concentration and OD260/OD280 of each DNA sample

SDS concentration	Concentration (ng/. Mu.L)	OD260/OD280
			0.2％	75.8	2.50
0.6％	83.2	2.64
			2.4％	129.5	2.83
3.2％	76.3	2.66

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Claims (10)

1. The crab meat DNA extraction kit comprises a lysate, and is characterized in that the lysate comprises SDS, proteinase K and RNase A.

2. The crab meat DNA extraction kit according to claim 1, wherein the lysate comprises 0.6-2.4% SDS, 2% proteinase K and 1% RNase A.

3. The crab meat DNA extraction kit of claim 1, wherein the lysate comprises 1% SDS, 2% proteinase K and 1% RNase A.

4. The crab meat DNA extraction kit of claim 1, further comprising a rinse solution and an eluent.

5. The crab meat DNA extraction kit of claim 4, wherein the rinse solution is an organic alcohol solution.

6. The crab meat DNA extraction kit of claim 4, wherein the eluent is selected from one of TE buffer, tris-HCl buffer and water.

7. A method for extracting crab meat DNA using a crab meat DNA extraction kit according to claim 1, comprising the steps of:

s1, adding the cracking liquid into a grinding pipe containing steel balls, taking a proper amount of crab meat into the grinding pipe, and grinding for 1-3 min by using a grinding instrument at 50 Hz; the method comprises the steps of carrying out a first treatment on the surface of the

S2, placing the grinding sample tube in a water bath kettle at 65 ℃ for 13-15 min;

s3, placing the grinding sample tube in a water bath kettle at 70 ℃ for 13-15 min;

s4, placing the grinding sample tube into a centrifugal machine 8000-10000 g to be centrifuged for 10-15 min, and transferring the supernatant into a new centrifugal tube;

s5, adding chloroform with the volume of 0.5 times, reversing and uniformly mixing until the solution is completely emulsified into white, and centrifuging for 8-15 min at the temperature of 12000-15000 Xg and the temperature of 4 ℃;

s6, sucking supernatant fluid and transferring the supernatant fluid into a new sterilizing centrifuge tube;

s7, adding magnetic beads with the volume of 0.6 times of that of the supernatant fluid, uniformly mixing by blowing, and standing at room temperature for 3-8 min;

s8, placing the centrifuge tube in a magnetic rack until the solution is clear, and sucking and discarding the supernatant;

s9, keeping the centrifuge tube on a magnetic rack, adding 200 mu L of rinsing liquid, standing at room temperature for 20-40S, and then sucking and discarding the supernatant;

s10, performing instantaneous centrifugation, discarding redundant rinsing liquid in the centrifuge tube, and drying the magnetic beads at room temperature for 2-5 min;

s11, taking down the centrifuge tube from the magnetic rack, and standing at room temperature for 2-5 min; adding 100 mu L of eluent, lightly blowing and mixing by a pipettor, and standing for 3-8 min at room temperature;

s12, placing the centrifuge tube on a magnetic rack, standing at room temperature for 3-8 min until the solution is clear, sucking the supernatant, and transferring the supernatant into a corresponding new sample storage tube to obtain purified crab meat DNA.

8. The method according to claim 7, characterized in that step S9 is repeated one time before step S10.

9. The method of claim 7, wherein the rinse solution is 80% ethanol.

10. The method of claim 7, wherein the eluent is selected from one of TE buffer, tris-HCl buffer and water.

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