CN112175944A - Method for extracting shellfish DNA from shellfish blood - Google Patents
Method for extracting shellfish DNA from shellfish blood Download PDFInfo
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- CN112175944A CN112175944A CN202011238577.8A CN202011238577A CN112175944A CN 112175944 A CN112175944 A CN 112175944A CN 202011238577 A CN202011238577 A CN 202011238577A CN 112175944 A CN112175944 A CN 112175944A
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Abstract
The invention discloses a method for extracting shellfish DNA from shellfish blood, which comprises the following steps: collecting shellfish blood and pretreating; step two: extracting DNA; the invention mainly aims to solve the problem that the shellfish parents die and the samples are lost due to the culture conditions or other problems in the shellfish breeding process; the traditional method needs destructive sampling for extracting shellfish DNA, so that the same shellfish can be sampled only in one growth stage, the invention can extract blood samples of shellfish in different growth stages, can extract DNA in different stages, and has important research significance and practical value in aspects of shellfish whole-gene selective breeding and the like, the experimental material of the invention is easier to obtain, and blood can be repeatedly taken for many times; compare with the tradition and used the animal tissue sample and reduced the process of grinding the appearance for the efficiency of extracting DNA promotes greatly.
Description
Technical Field
The invention relates to the field of DNA extraction, in particular to a method for extracting shellfish DNA from shellfish blood.
Background
The shellfish farming industry plays an important role in supplying high-quality protein, increasing fisherman income, improving offshore environment and the like. Meanwhile, shellfish culture also faces the problem of resource failure, more and more scholars obtain shellfish DNA through molecular biology technology and research the genetic information thereof, and the molecular assisted breeding method is used for achieving the purposes of improving shellfish quality and breeding shellfish stress-resistant new products and solving the problem of shellfish resource exhaustion. However, the traditional sampling methods mostly focus on either destructive sampling or non-destructive sampling, such as direct dissection, opening the shell by using a mouth expander, etc., which causes strong mechanical damage and even death to the shellfish, and is not beneficial to the later-stage seed preservation of the shellfish, genetic breeding and the protection of shellfish resources, especially to endangered precious shellfish. Therefore, the method is used for extracting shellfish DNA from shellfish blood.
Disclosure of Invention
The invention aims to provide a method for extracting shellfish DNA from shellfish blood to solve the problems of the background technology.
In order to achieve the purpose, the invention provides the following technical scheme: a method for extracting shellfish DNA from shellfish blood comprises the following steps:
the method comprises the following steps: collecting shellfish blood and pretreating; opening a small hole at the thick adductor muscle of the shellfish, and extracting blood of the shellfish by using a disposable sterile syringe; the collected blood was transferred to a 1.5mL centrifuge tube, mixed and centrifuged, and the supernatant was collected.
Step two: extracting DNA;
s21: putting the blood extracted in the step one into a 1.5mL centrifuge tube, adding 600 mu L of lysate and 10 mu L of proteinase K, taking 2 15mL centrifuge tubes, respectively adding 10mL of lysate and 10 mu L of proteinase K into each centrifuge tube, uniformly mixing by using a gun head, adding 610 mixed liquor into each sample, and digesting at 55 ℃ overnight;
s22: adding chloroform and phenol into 2 10mL large centrifuge tubes respectively, and adding 300 mu L phenol and chloroform into each sample respectively;
s23: reversing and mixing evenly, centrifuging, sucking 500 mu L of supernatant fluid tightly attached to the liquid surface, and adding 1mL of absolute ethyl alcohol for precipitation;
s24: fully and uniformly mixing the samples, and centrifuging after finding floccules;
s25: pouring off waste liquid, adding 500 μ L70% ethanol, reversing back and forth, mixing, centrifuging, pouring off waste liquid, opening cover, air drying for 10min, and dissolving at room temperature.
As a preferable technical scheme of the invention, the diameter of the small hole in the step one is 0.3-0.4 mm.
As a preferred technical scheme of the invention, the centrifugal rotating speed in the step one is 800 r/min; the time is 3 min.
As a preferable technical scheme of the invention, the centrifugal rotating speeds in S23 and S24 are both 1200 r/min; the time is 10 min.
As a preferable technical scheme of the invention, the centrifugal rotating speed in S25 is 1200 r/min; the time is 5 min.
The invention has the beneficial effects that: the invention mainly aims to solve the problem that the shellfish parents die and the samples are lost due to the culture conditions or other problems in the shellfish breeding process; the traditional method needs destructive sampling for extracting shellfish DNA, so that the same shellfish can be sampled only in one growth stage of the shellfish.
Compared with the traditional method, the method has the advantages that experimental materials are easier to obtain, and blood can be repeatedly taken for many times (the safe blood taking amount does not influence the growth and development of the shellfish); compared with the traditional animal tissue sampling, the method reduces the sample grinding process, greatly improves the efficiency of extracting DNA, and can improve the efficiency by about 40%; the use of blood to extract DNA can secure animals without the need for destructive sampling.
Drawings
FIG. 1 is a flow chart of the method of the present invention.
Detailed Description
The following detailed description of the preferred embodiments of the present invention, taken in conjunction with the accompanying drawings, will make the advantages and features of the invention more readily understood by those skilled in the art, and thus will more clearly and distinctly define the scope of the invention.
Example (b): referring to fig. 1, the present invention provides a technical solution: a method for extracting shellfish DNA from shellfish blood comprises the following steps:
the method comprises the following steps: collecting shellfish blood and pretreating; opening a small hole of 0.3-0.4mm at the thick adductor muscle of the shellfish, and extracting blood of the shellfish by using a disposable sterile syringe; transferring the collected blood to a 1.5mL centrifuge tube, uniformly mixing, centrifuging at 800r/min for 3min, and taking supernatant;
step two: extracting DNA;
s21: putting the blood extracted in the step one into a 1.5mL centrifuge tube, adding 600 mu L of lysate and 10 mu L of proteinase K, taking 2 15mL centrifuge tubes, respectively adding 10mL of lysate and 10 mu L of proteinase K into each centrifuge tube, uniformly mixing by using a gun head, adding 610 mixed liquor into each sample, and digesting at 55 ℃ overnight;
s22: adding chloroform and phenol into 2 10mL large centrifuge tubes respectively, and adding 300 mu L phenol and chloroform into each sample respectively;
s23: reversing back and forth, mixing, centrifuging at 1200r/min for 10 min; sucking 500 mu L of supernatant liquid tightly close to the liquid surface, and adding 1mL of absolute ethyl alcohol for precipitation;
s24: fully and uniformly mixing the sample, and centrifuging at 1200r/min for 10min when floccules are found;
s25: pouring off waste liquid, adding 500 μ L70% ethanol, reversing and mixing, centrifuging at 1200r/min for 5min, pouring off waste liquid, opening cover and air drying for 10 min; dissolving at room temperature.
The invention mainly aims to solve the problem that the shellfish parents die and the samples are lost due to the culture conditions or other problems in the shellfish breeding process; the traditional method needs destructive sampling for extracting shellfish DNA, so that the same shellfish can be sampled only in one growth stage of the shellfish.
Compared with the traditional method, the method has the advantages that experimental materials are easier to obtain, and blood can be repeatedly taken for many times (the safe blood taking amount does not influence the growth and development of the shellfish); compared with the traditional animal tissue sampling, the method reduces the sample grinding process, greatly improves the efficiency of extracting DNA, and can improve the efficiency by about 40%; the use of blood to extract DNA can secure animals without the need for destructive sampling.
The above examples only show some embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.
Claims (5)
1. A method for extracting shellfish DNA from shellfish blood is characterized in that: the method comprises the following specific steps:
the method comprises the following steps: collecting shellfish blood and pretreating; opening a small hole at the thick adductor muscle of the shellfish, and extracting blood of the shellfish by using a disposable sterile syringe; the collected blood was transferred to a 1.5mL centrifuge tube, mixed and centrifuged, and the supernatant was collected.
Step two: extracting DNA;
s21: putting the blood extracted in the step one into a 1.5mL centrifuge tube, adding 600 mu L of lysate and 10 mu L of proteinase K, taking 2 15mL centrifuge tubes, respectively adding 10mL of lysate and 10 mu L of proteinase K into each centrifuge tube, uniformly mixing by using a gun head, adding 610 mixed liquor into each sample, and digesting at 55 ℃ overnight;
s22: adding chloroform and phenol into 2 10mL large centrifuge tubes respectively, and adding 300 mu L phenol and chloroform into each sample respectively;
s23: reversing and mixing evenly, centrifuging, sucking 500 mu L of supernatant fluid tightly attached to the liquid surface, and adding 1mL of absolute ethyl alcohol for precipitation;
s24: fully and uniformly mixing the samples, and centrifuging after finding floccules;
s25: pouring off waste liquid, adding 500 μ L70% ethanol, reversing back and forth, mixing, centrifuging, pouring off waste liquid, opening cover, air drying for 10min, and dissolving at room temperature.
2. The method for extracting shellfish DNA from shellfish blood as claimed in claim 1, wherein: the diameter of the small hole in the step one is 0.3-0.4 mm.
3. The method for extracting shellfish DNA from shellfish blood as claimed in claim 1, wherein: the centrifugal rotating speed in the first step is 800 r/min; the time is 3 min.
4. The method for extracting shellfish DNA from shellfish blood as claimed in claim 1, wherein: the centrifugal rotating speeds in the S23 and the S24 are both 1200 r/min; the time is 10 min.
5. The method for extracting shellfish DNA from shellfish blood as claimed in claim 1, wherein: the centrifugal rotating speed in the S25 is 1200 r/min; the time is 5 min.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202011238577.8A CN112175944A (en) | 2020-11-09 | 2020-11-09 | Method for extracting shellfish DNA from shellfish blood |
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| CN202011238577.8A CN112175944A (en) | 2020-11-09 | 2020-11-09 | Method for extracting shellfish DNA from shellfish blood |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103320425A (en) * | 2013-07-02 | 2013-09-25 | 中国海洋大学 | Rapid extraction method of shellfish deoxyribonucleic acid (DNA) for large-scale and high-throughput genotyping |
| CN103756999A (en) * | 2014-02-17 | 2014-04-30 | 中国水产科学研究院黑龙江水产研究所 | Method for quickly extracting genome DNA from fish blood |
| CN107513528A (en) * | 2017-10-16 | 2017-12-26 | 鲁东大学 | A kind of pen shell shell DNA extraction method, identification primer and kit |
| CN110343742A (en) * | 2019-07-23 | 2019-10-18 | 中国海洋大学 | A kind of micro shellfish DNA extraction method for high-throughput sequencing library preparation |
| CN110794024A (en) * | 2019-11-30 | 2020-02-14 | 中国海洋大学 | Standardized determination method for shellfish blood physiological indexes |
-
2020
- 2020-11-09 CN CN202011238577.8A patent/CN112175944A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103320425A (en) * | 2013-07-02 | 2013-09-25 | 中国海洋大学 | Rapid extraction method of shellfish deoxyribonucleic acid (DNA) for large-scale and high-throughput genotyping |
| CN103756999A (en) * | 2014-02-17 | 2014-04-30 | 中国水产科学研究院黑龙江水产研究所 | Method for quickly extracting genome DNA from fish blood |
| CN107513528A (en) * | 2017-10-16 | 2017-12-26 | 鲁东大学 | A kind of pen shell shell DNA extraction method, identification primer and kit |
| CN110343742A (en) * | 2019-07-23 | 2019-10-18 | 中国海洋大学 | A kind of micro shellfish DNA extraction method for high-throughput sequencing library preparation |
| CN110794024A (en) * | 2019-11-30 | 2020-02-14 | 中国海洋大学 | Standardized determination method for shellfish blood physiological indexes |
Non-Patent Citations (1)
| Title |
|---|
| 常亚青主编: "《贝类增养殖学》", 31 August 2008, 北京:中国农业出版社 * |
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