CN111321231A - Method for molecularly identifying gynogenesis progeny of rainbow trout - Google Patents
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Abstract
The invention provides a method for molecularly identifying gynogenesis offspring of rainbow trout, which comprises the following steps: selecting tail fins of gynogenesis offspring of the American redfish and the rainbow trout to extract genome DNA, and identifying the gynogenesis offspring condition of the rainbow trout by the marking verification of whether a lane on a display strip contains a male parent or not after a PCR reaction system and a program, PCR product agarose gel electrophoresis detection, polyacrylamide gel electrophoresis and silver staining. According to the invention, the screened molecular marker AOMM108 is used for screening gynogenesis offspring of the rainbow trout, so that the gynogenesis offspring can be identified earlier from the young fish stage of the rainbow trout, the economic loss caused in the later breeding stage is reduced, and the breeding effect is improved. The method is simple and rapid, has high accuracy, and can play an important role in the field of genetic breeding of rainbow trout.
Description
Technical Field
The invention belongs to the technical field of aquatic product genetic breeding, and particularly relates to a method for molecularly identifying gynogenesis progeny of rainbow trout.
Background
The fish gynogenesis technology has an extremely important application value in the field of aquaculture, and the artificial induction of the fish gynogenesis is a special sexual reproduction mode of a filial generation by using genetically inactivated sperms to activate ova and then inhibiting polar body elimination or first cleavage of fertilized ova. The male fish of rainbow trout generally reaches sexual maturity at 2 ages, and the female fish reaches sexual maturity at 3 ages. Mature individuals show the characteristics of reduced growth rate, improved mortality rate, poorer meat quality and appearance and the like, and are unfavorable for culture production. The sperms of the American red-spotted salmon are adopted to carry out heterologous sperm induction on the gynogenesis of the rainbow trout, all the obtained offspring are the female fish population, the adverse effect can be reduced, and meanwhile, breeding materials can be provided for the next breeding work. However, the external morphology of the experimental offspring is difficult to distinguish whether the experimental offspring is the true gynogenesis offspring, and particularly in the juvenile fish stage, an effective means for accurately identifying the gynogenesis offspring of the rainbow trout does not exist at present. As the individuals of the filial generations are sterile, great waste is caused to the breeding work, which is a difficult problem in the breeding work of the rainbow trout, and therefore, the early, high-efficiency and accurate identification of the gynogenesis progeny of the rainbow trout is a core problem to be solved.
Disclosure of Invention
The invention aims to solve the technical problem in the prior art and provides a method for identifying gynogenesis progeny of rainbow trout by molecules.
The technical scheme is as follows for solving the technical problem of the invention:
a method for molecularly identifying gynogenesis offspring of rainbow trout comprises the following steps: selecting tail fins of gynogenesis offspring of the American redfish and the rainbow trout to extract genome DNA, and identifying the gynogenesis offspring condition of the rainbow trout by the marking verification of whether a lane on a display strip contains a male parent or not after a PCR reaction system and a program, PCR product agarose gel electrophoresis detection, polyacrylamide gel electrophoresis and silver staining;
wherein the primer in the PCR reaction system is AOMM108,
F:GGAGGCAGCTCCTGTTTTC，R:GCTGCGCCATCTCTCTATCT。
the PCR reaction system is as follows: the total was 13. mu.L, with 6.5. mu.L of PCR premix enzyme at 2-fold concentration;TaqDNA polymerase: 0.1U/ML; magnesium chloride: 4 mmol/L; dNTP mix: 0.4mmol/L and 0.5. mu.L of each of two primers of 10 mmol/L; 0.5. mu.L of DNA template; the rest double distilled water is complemented;
the PCR reaction program is as follows: pre-denaturation at 94 ℃ for 3 min; pre-denaturation at 94 ℃ for 35 s, annealing at 59 ℃ for 45 s, and extension at 72 ℃ for 45 s for 30 cycles; after the reaction was complete, the extension was carried out for a further 8 min at 72 ℃.
The method for identifying the gynogenesis progeny of the rainbow trout by the molecules comprises the following specific steps:
(1) DNA extraction and detection
Collecting tail fin samples of gynogenesis offspring of the American redspot salmon and the rainbow trout, and extracting genome DNA by adopting a high-salt concentration extraction method;
(2) design of primers
1 pair of primers is AOMM108, F: GGAGGCAGCTCCTGTTTTCT, R: GCTGCGCCATCTCTCTATCT;
(3) PCR reaction system and program
Amplifying the target fragment by using the synthesized upstream and downstream primers, wherein the PCR reaction system comprises: the total volume is 13 μ L, including 13 μ L total volume, wherein the PCR premix enzyme is 6.5 μ L at 2-fold concentration; TaqDNA polymerase: 0.1U/ML; magnesium chloride: 4 mmol/L; dNTP mix: 0.4mmol/L and 0.5. mu.L of each of two primers of 10 mmol/L; 0.5. mu.L of DNA template; the rest double distilled water is complemented;
the PCR reaction program is as follows: pre-denaturation at 94 ℃ for 3 min; pre-denaturation at 94 ℃ for 35 s, annealing at 59 ℃ for 45 s, and extension at 72 ℃ for 45 s for 30 cycles; after the reaction is finished, the extension is carried out for 8 min at 72 ℃;
(4) and detecting the PCR product by agarose gel electrophoresis
Performing electrophoresis on the amplification product in agarose gel with the mass fraction of 10% and 1 × TAE buffer solution at the voltage of 5V/cm for 15min, and detecting whether a strip exists;
(5) polyacrylamide gel electrophoresis and silver staining
Preparing gel by using double distilled water, acrylamide, 5 × TBE, Tetramethylethylenediamine (TEMED) and AP, injecting the prepared gel into a gel tank, inserting a comb, standing and solidifying, then taking down the comb, assembling the comb on an electrophoresis tank, loading a product, after electrophoresis is finished, taking out the gel, putting the gel into a porcelain plate filled with pure water, adding silver nitrate after cleaning once, placing the porcelain plate on a shaking table for dyeing, pouring off the silver nitrate, adding pure water for washing, adding NaOH solution into a magnetic disc, placing the magnetic disc on the shaking table for dyeing, starting presenting strips, pouring off the NaOH solution, adding pure water for washing, flatly paving a film on a viewing box, and photographing and recording;
(6) and mark verification
The result shows that the lanes of the gynogenesis offspring do not contain the strip of the male parent, the genetic material of the gynogenesis offspring can be identified to be from the female parent, and the genetic material infiltration of the male parent is not found; the result shows that the lane of the gynogenesis offspring contains the band of the male parent, and the gynogenesis offspring can be identified as the filial offspring.
The primer in the step (2) is designed as follows: based on the sequence information of accession number AF352758.1 in GenBank database, 1 pair of primers AOMM108, F: GGAGGCAGCTCCTGTTTTCT, R: GCTGCGCCATCTCTCTATCT, synthesized by Shanghai Bioengineering technology services, Inc., were designed using Primer3, v.0.4.0.
The electrophoresis conditions in the step (5) are that the electrophoresis buffer solution is 1 × TBE, the voltage is 200V, and the electrophoresis time is 2.5-3 h.
The specific method for extracting and detecting the DNA in the step (1) is as follows:
a. digestion of
Taking 0.5-1g tail fin sample of gynogenesis offspring of the American red spot salmon and the rainbow trout, adding the tail fin sample into a centrifuge tube, adding 500ul HomBuffer and 15ul proteinase K, putting the centrifuge tube into a water bath kettle at 55-60 ℃ for 2-3h, and shaking up once every 30 minutes;
b. by extraction of
Adding 500ul of NaCL and 4.5mol/L of chloroform into a centrifuge tube, continuously shaking for 15min, then centrifuging for 10min, and absorbing 800ul of supernate into a new centrifuge tube after centrifuging;
c. precipitating and drying
Adding 595ul of isopropanol into a new centrifugal tube, uniformly mixing for 1-2 minutes, placing at-20 ℃ for precipitation for 2 hours, centrifuging at 12000 and 13000rpm for 10 minutes, removing supernatant, adding 500ul of ethanol with volume fraction of 70 percent, washing for 5 minutes, centrifuging for 10 minutes, removing supernatant, placing in a sterile ultra-clean bench for natural drying, adding 60ul of sterile double distilled water into the dried centrifugal tube, fully dissolving DNA, and storing at-20 ℃ for later use;
the centrifugation speed in the step b and the step c is 12000-13000 rpm/min.
Taking 25.6ml of double distilled water, 18.4ml of 30% acrylamide by mass, 11ml of 5 × TBE, 0.134ml of tetramethylethylenediamine TEMED and 10% AP350ul by mass, uniformly stirring to prepare gel, injecting the prepared gel into an assembled gel tank, inserting a comb, standing for 2-3h for solidification, taking down the comb, assembling the comb onto an electrophoresis tank, wherein the sample loading amount of a PCR product is 5 mu L, taking out the gel after electrophoresis, putting the gel into a porcelain dish filled with pure water, adding 800ml of silver nitrate with the mass fraction of 1% after washing once, putting the porcelain dish on a shaking table for dyeing for 8-10 minutes, pouring the silver nitrate, adding the pure water, quickly washing for 10s, adding 1000ml of NaOH solution with the mass fraction of 2% into the magnetic dish, putting the porcelain dish on the shaking table for dyeing for 10 minutes, beginning to present strips, pouring the NaOH solution, adding the pure water for washing, spreading the film on a film box, and recording the pictures.
The invention has the beneficial effects that: by applying the screened molecular marker AOMM108 to screen gynogenesis offspring of the rainbow trout, the gynogenesis offspring can be identified earlier from the young fish stage of the rainbow trout, the economic loss caused in the later breeding stage is reduced, and the breeding effect is improved. The method is simple and rapid, has high accuracy, and can play an important role in the field of genetic breeding of rainbow trout.
Drawings
FIG. 1 is an amplification map of gynogenesis progeny and parents thereof in the microsatellite molecular marker AF 352758.1; gynogenesis of individuals: lanes 1-13, M: marker, male parent: M14-M17, female parent: F18-F21.
Detailed Description
The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.
Example 1
A method for molecularly identifying gynogenesis offspring of rainbow trout comprises the following specific steps:
1. extraction and detection of DNA
Collecting samples, taking 0.5 g of tail fin samples of gynogenesis filial generations of the American red spotted salmon and the rainbow trout, and extracting genome DNA by adopting a high-salt concentration extraction method.
1.1 digestion
Adding the tail fin tissue into a centrifuge tube, adding 500ul of Hom Buffer and 15ul of protease K, putting into a water bath kettle at 60 ℃ for 3h, and shaking up once every 30 minutes;
1.2 extraction of
Adding 4.5mol/L NaCL500ul and 300ul chloroform into a centrifuge tube, shaking up for 15min, centrifuging at 12000rpm/min for 10min, and sucking 800ul supernatant into a new centrifuge tube after centrifugation;
1.3 precipitation and drying
595ul of isopropanol is added, mixed evenly for 1 minute, placed at minus 20 ℃ for precipitation for 2 hours, centrifuged at 13000rpm/min for 10 minutes, the supernatant is removed, 500ul of ethanol with 70 percent volume fraction is added for washing for 5 minutes, centrifuged at 12000rpm/min for 10 minutes, the supernatant is removed, placed in a sterile super clean bench for natural drying, 60ul of sterile double distilled water is added into an air-dried centrifugal tube, and the DNA is stored at minus 20 ℃ for standby after being fully dissolved.
2. Primer design
Based on the sequence information of accession No. AF352758.1 in GenBank database, 1 pair of primers AOMM108(F: GGAGGCAGCTCCTGTTTTCT, R: GCTGCGCCATCTCTCTATCT) was designed using Primer3 (v.0.4.0) and synthesized by Shanghai Bioengineering technology services, Inc.
3. PCR reaction system and program
PCR reaction system of 13. mu.L including 6.5. mu.L of dye-containing 2 × Taq PCR Mastermix (TaqDNA polymerase: 0.1U/ML; MgCl2: 4 mmol/L; dNTPseach:0.4 mmol/L), 0.5. mu.L of two primers (10 mmol/L), 0.5. mu.L of DNA template, and the balance of double distilled water. The PCR reaction program is: pre-denaturation at 94 ℃ for 3 min; pre-denaturation at 94 ℃ for 35 s, annealing at 59 ℃ for 45 s, and extension at 72 ℃ for 45 s for 30 cycles; after the reaction was complete, the extension was carried out for a further 8 min at 72 ℃.
4. Agarose gel electrophoresis detection of PCR products
The amplification product was electrophoresed in 10% by mass agarose gel and 1 × TAE buffer at 5V/cm for 15min to detect the presence of bands.
5. Polyacrylamide gel electrophoresis and silver staining
Taking 25.6ml of double distilled water, 18.4ml of acrylamide with the mass fraction of 30%, 11ml of 5 × TBE, 0.134ml of TEMED and 350ul of AP with the mass fraction of 10%, fully and uniformly stirring by using a glass rod, injecting the prepared gel into an assembled gel tank, inserting a comb, standing for 3 hours for solidification, taking down the comb, assembling the comb on an electrophoresis tank, setting the sample loading amount of a PCR product to be 5 mu L, and carrying out electrophoresis under the electrophoresis conditions that an electrophoresis buffer solution is 1 × TBE, the voltage is 200V, the electrophoresis time is 3 hours, taking out the gel, putting the gel into a porcelain dish filled with pure water, quickly cleaning once, adding 800ml of silver nitrate with the mass fraction of 1%, placing the porcelain dish on a shaking table for dyeing for 8 minutes, pouring off the silver nitrate, adding the pure water, quickly washing for 10 seconds, adding 1000ml of NaOH solution with the mass fraction of 2% into the magnetic dish, placing the porcelain dish on the shaking table for dyeing for 10 minutes, starting to present strips, pouring off the NaOH solution, adding the washing water, placing the film on a film box for photographing, and.
6. Mark verification
The result shows that the lanes of the gynogenesis offspring do not contain the strip of the male parent, the genetic material of the gynogenesis offspring can be identified to be from the female parent, and the genetic material infiltration of the male parent is not found; the result shows that the lane of the gynogenesis offspring contains the band of the male parent, and the gynogenesis offspring can be identified as the filial offspring.
Example 2
A method for molecularly identifying gynogenesis offspring of rainbow trout comprises the following specific steps:
1. extraction and detection of DNA
Collecting samples, taking 1g of tail fin samples of gynogenesis offspring of the American red-spotted salmon and the rainbow trout, and extracting genome DNA by a high-salt concentration extraction method.
1.1 digestion
Adding the tail fin tissue into a centrifuge tube, adding 500ul of Hom Buffer and 15ul of protease K, putting into a water bath kettle at 55 ℃ for 2h, and shaking up once every 30 minutes;
1.2 extraction of
Adding 4.5mol/L NaCL500ul and 300ul chloroform into a centrifuge tube, shaking up for 15min, centrifuging at the rotation speed of 13000rpm/min for 10min, and sucking 800ul supernatant into a new centrifuge tube after centrifugation;
1.3 precipitation and drying
595ul of isopropanol is added, mixed evenly for 2 minutes, placed at minus 20 ℃ for precipitation for 2 hours, centrifuged at 12000rpm/min for 10 minutes, the supernatant is removed, 500ul of ethanol with volume fraction of 70% is added for washing for 5 minutes, centrifuged at 13000rpm/min for 10 minutes, the supernatant is removed, placed in a sterile super clean bench for natural drying, 60ul of sterile double distilled water is added into an air-dried centrifugal tube, and the DNA is stored at minus 20 ℃ for standby after being fully dissolved.
2. Primer design
Based on the sequence information of accession No. AF352758.1 in GenBank database, 1 pair of primers AOMM108(F: GGAGGCAGCTCCTGTTTTCT, R: GCTGCGCCATCTCTCTATCT) was designed using Primer3 (v.0.4.0) and synthesized by Shanghai Bioengineering technology services, Inc.
3. PCR reaction system and program
PCR reaction system of 13. mu.L including 6.5. mu.L of dye-containing 2 × Taq PCR Mastermix (TaqDNA polymerase: 0.1U/ML; MgCl2: 4 mmol/L; 0.4mmol/L of dNTPseach), 0.5 mu L of two primers (10 mmol/L), 0.5 mu L of DNA template and the balance of double distilled water. The PCR reaction program is: pre-denaturation at 94 ℃ for 3 min; pre-denaturation at 94 ℃ for 35 s, annealing at 59 ℃ for 45 s, and extension at 72 ℃ for 45 s for 30 cycles; after the reaction was complete, the extension was carried out for a further 8 min at 72 ℃.
4. Agarose gel electrophoresis detection of PCR products
The amplification product was electrophoresed in 10% by mass agarose gel and 1 × TAE buffer at 5V/cm for 15min to detect the presence of bands.
5. Polyacrylamide gel electrophoresis and silver staining
Taking 25.6ml of double distilled water, 18.4ml of acrylamide with the mass fraction of 30%, 11ml of 5 × TBE, 0.134ml of TEMED and 350ul of AP with the mass fraction of 10%, fully and uniformly stirring by using a glass rod, injecting the prepared gel into an assembled gel tank, inserting a comb, standing for 2 hours for solidification, taking down the comb, assembling the comb on an electrophoresis tank, setting the sample loading amount of a PCR product to be 5 mu L, and carrying out electrophoresis under the electrophoresis conditions that an electrophoresis buffer solution is 1 × TBE, the voltage is 200V, the electrophoresis time is 2.5 hours, taking out the gel, putting the gel into a porcelain dish filled with pure water, quickly cleaning once, adding 800ml of silver nitrate with the mass fraction of 1%, placing the porcelain dish on a shaking table for dyeing for 10 minutes, pouring off the silver nitrate, adding the pure water, quickly washing for 10 seconds, adding 1000ml of NaOH solution with the mass fraction of 2% into the magnetic dish, placing the porcelain dish on the shaking table for dyeing for 10 minutes, beginning to present strips, pouring off the NaOH solution, adding the NaOH solution, washing, laying the film on a film box, and.
6. Mark verification
The result shows that the lanes of the gynogenesis offspring do not contain the strip of the male parent, the genetic material of the gynogenesis offspring can be identified to be from the female parent, and the genetic material infiltration of the male parent is not found; the result shows that the lane of the gynogenesis offspring contains the band of the male parent, and the gynogenesis offspring can be identified as the filial offspring.
Claims (8)
1. A method for molecularly identifying gynogenesis offspring of rainbow trout is characterized by comprising the following steps: selecting tail fins of gynogenesis offspring of the American redfish and the rainbow trout to extract genome DNA, and identifying the gynogenesis offspring condition of the rainbow trout by the marking verification of whether a lane on a display strip contains a male parent or not after a PCR reaction system and a program, PCR product agarose gel electrophoresis detection, polyacrylamide gel electrophoresis and silver staining;
wherein the primer in the PCR reaction system is AOMM108,
F:GGAGGCAGCTCCTGTTTTC，R:GCTGCGCCATCTCTCTATCT。
2. the method of claim 1, wherein said method comprises the steps of:
the PCR reaction system is as follows: the total was 13. mu.L, with 6.5. mu.L of PCR premix enzyme at 2-fold concentration;TaqDNA polymerase: 0.1U/ML; magnesium chloride: 4 mmol/L; dNTP mix: 0.4mmol/L and 0.5. mu.L of each of two primers of 10 mmol/L; 0.5. mu.L of DNA template; the rest double distilled water is complemented;
the PCR reaction program is as follows: pre-denaturation at 94 ℃ for 3 min; pre-denaturation at 94 ℃ for 35 s, annealing at 59 ℃ for 45 s, and extension at 72 ℃ for 45 s for 30 cycles; after the reaction was complete, the extension was carried out for a further 8 min at 72 ℃.
3. The method for molecularly identifying gynogenesis progeny of rainbow trout according to claim 1 or 2, which is characterized by comprising the following steps:
(1) DNA extraction and detection
Collecting tail fin samples of gynogenesis offspring of the American redspot salmon and the rainbow trout, and extracting genome DNA by adopting a high-salt concentration extraction method;
(2) design of primers
1 pair of primers is AOMM108, F: GGAGGCAGCTCCTGTTTTCT, R: GCTGCGCCATCTCTCTATCT;
(3) PCR reaction system and program
Amplifying the target fragment by using the synthesized upstream and downstream primers, wherein the PCR reaction system comprises: the total volume is 13 μ L, including 13 μ L total volume, wherein the PCR premix enzyme is 6.5 μ L at 2-fold concentration; TaqDNA polymerase: 0.1U/ML; magnesium chloride: 4 mmol/L; dNTP mix: 0.4mmol/L and 0.5. mu.L of each of two primers of 10 mmol/L; 0.5. mu.L of DNA template; the rest double distilled water is complemented;
the PCR reaction program is as follows: pre-denaturation at 94 ℃ for 3 min; pre-denaturation at 94 ℃ for 35 s, annealing at 59 ℃ for 45 s, and extension at 72 ℃ for 45 s for 30 cycles; after the reaction is finished, the extension is carried out for 8 min at 72 ℃;
(4) and detecting the PCR product by agarose gel electrophoresis
Performing electrophoresis on the amplification product in agarose gel with the mass fraction of 10% and 1 × TAE buffer solution at the voltage of 5V/cm for 15min, and detecting whether a strip exists;
(5) polyacrylamide gel electrophoresis and silver staining
Preparing gel by using double distilled water, acrylamide, 5 × TBE, Tetramethylethylenediamine (TEMED) and AP, injecting the prepared gel into a gel tank, inserting a comb, standing and solidifying, then taking down the comb, assembling the comb on an electrophoresis tank, loading a product, after electrophoresis is finished, taking out the gel, putting the gel into a porcelain plate filled with pure water, adding silver nitrate after cleaning once, placing the porcelain plate on a shaking table for dyeing, pouring off the silver nitrate, adding pure water for washing, adding NaOH solution into a magnetic disc, placing the magnetic disc on the shaking table for dyeing, starting presenting strips, pouring off the NaOH solution, adding pure water for washing, flatly paving a film on a viewing box, and photographing and recording;
(6) and mark verification
The result shows that the lanes of the gynogenesis offspring do not contain the strip of the male parent, the genetic material of the gynogenesis offspring can be identified to be from the female parent, and the genetic material infiltration of the male parent is not found; the result shows that the lane of the gynogenesis offspring contains the band of the male parent, and the gynogenesis offspring can be identified as the filial offspring.
4. The method of claim 3, wherein said method comprises the steps of: the primer in the step (2) is designed as follows: based on the sequence information of accession number AF352758.1 in GenBank database, 1 pair of primers AOMM108, F: GGAGGCAGCTCCTGTTTTCT, R: GCTGCGCCATCTCTCTATCT, synthesized by Shanghai Bioengineering technology services, Inc., were designed using Primer3, v.0.4.0.
5. The method for molecularly identifying gynogenesis progeny of rainbow trout according to claim 4, wherein the electrophoresis conditions in step (5) are electrophoresis buffer of 1 × TBE, voltage of 200V and electrophoresis time of 2.5-3 h.
6. The method for molecularly identifying gynogenesis progeny of rainbow trout according to claim 3 or 5, wherein the specific method for extracting and detecting the DNA in the step (1) is as follows:
a. digestion of
Taking 0.5-1g tail fin sample of gynogenesis offspring of the American red spot salmon and the rainbow trout, adding the tail fin sample into a centrifuge tube, adding 500ul HomBuffer and 15ul proteinase K, putting the centrifuge tube into a water bath kettle at 55-60 ℃ for 2-3h, and shaking up once every 30 minutes;
b. by extraction of
Adding 500ul of NaCL and 4.5mol/L of chloroform into a centrifuge tube, continuously shaking for 15min, then centrifuging for 10min, and absorbing 800ul of supernate into a new centrifuge tube after centrifuging;
c. precipitating and drying
595ul of isopropanol is added into a new centrifugal tube, mixed evenly for 1-2 minutes, placed at-20 ℃ for precipitation for 2 hours, centrifuged at 12000 and 13000rpm for 10 minutes, supernatant is removed, 500ul of ethanol with volume fraction of 70 percent is added for washing for 5 minutes, centrifuged for 10 minutes, supernatant is removed, placed in a sterile ultra-clean bench for natural drying, 60ul of sterile double distilled water is added into the dried centrifugal tube, DNA is fully dissolved, and then stored at-20 ℃ for standby.
7. The method of claim 6, wherein said method comprises the steps of: the centrifugation speed in the step b and the step c is 12000-13000 rpm/min.
8. The method for molecularly identifying the gynogenesis progeny of the rainbow trout according to claim 3 or 7, characterized in that the polyacrylamide gel electrophoresis and silver staining of step (5) are specifically carried out by taking 25.6ml of double distilled water, 18.4ml of acrylamide with the mass fraction of 30%, 11ml of 5 × TBE, 0.134ml of tetramethylethylenediamine TEMED and 10% of AP350ul, stirring uniformly to prepare gel, injecting the prepared gel into an assembled gel tank, inserting a comb, standing for 2-3h for solidification, removing the comb, assembling the comb on an electrophoresis tank, taking the PCR product with the sample amount of 5 μ L, taking out the gel after electrophoresis, placing the gel into a porcelain plate filled with pure water, adding 800ml of silver nitrate with the mass fraction of 1% after washing once, placing the porcelain plate on a shaking table for staining for 8-10 min, pouring the silver nitrate, adding pure water, washing for 10s quickly, adding 1000ml of NaOH solution with the mass fraction of 2% into the magnetic plate, placing the porcelain plate on the shaking table for staining for 10min, pouring out the silver nitrate, taking a picture, and recording the film on a plain film box.
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Cited By (2)
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|---|---|---|---|---|
| CN113699256A (en) * | 2021-09-26 | 2021-11-26 | 浙江省农业科学院 | Functional marker for rapidly identifying gynogenesis progeny of opsariichthys bidens and fancy carps and application of functional marker |
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| CN116064824A (en) * | 2022-07-06 | 2023-05-05 | 中国水产科学研究院黑龙江水产研究所 | High-temperature-resistant SNP molecular marker for rainbow trout and application thereof |
| CN116064824B (en) * | 2022-07-06 | 2024-06-21 | 中国水产科学研究院黑龙江水产研究所 | High-temperature-resistant SNP molecular marker for rainbow trout and application thereof |
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