SNP marker for Konosirus punctatus, and screening method and application thereof
A screening method and a technique for spot sting, applied in the field of molecular biology, can solve the problems of low acquisition purity of target DNA, complicated sequencing procedures, etc., and achieve the effect of reducing the cost of tag sequencing, improving the purity, and screening and detecting at low cost.
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Embodiment 1
[0036] A method for screening SNP markers of catfish, comprising the following steps:
[0037] Genomic DNA extraction: Cut the muscle or fin ray tissue samples of the spotted catfish into pieces, and place them in the extraction solution I: 70ml of Tris-HCl and disodium hydrogen phosphate with a concentration of 150mM, 0.5g of fatty alcohol ether ammonium sulfate, and 30ml of chlorine with a concentration of 130mM. Sodium chloride solution; add 0.5 μg plasmid DNA and 0.1 μg chitin, freeze and thaw 2-3 times, the freezing temperature is -166°C, the melting temperature is 50°C, centrifuge, collect the supernatant and add extract II: 10ml Tris-HCl with a concentration of 100mM and disodium hydrogen phosphate and 30ml of cationic polyacrylamide with a concentration of 1.6ppm were mixed upside down and centrifuged to produce a white precipitate, which was washed to obtain a DNA sample for use; circular DNA and chitin The special existence, on the one hand, can promote the combinati...
Embodiment 2
[0063]Circular DNA and chitin were not added in the process of extracting genomic DNA, and the rest were exactly the same as in Example 1; 28,730 SNPs were obtained by final filtration and screening, which indicated that the addition of circular DNA and chitin could effectively improve the integrity of the extracted DNA , which is beneficial to obtain the whole genome information of the catfish, and improve the accuracy and acquisition rate of SNP marker screening.
Embodiment 3
[0065] In the process of RAD library construction and high-throughput sequencing, chitin fluid was added during the PCR amplification system, the mass percentage of chitin was 0.16%, and the solvent was L-amino acid ethyl ester; the rest were completely consistent with Example 1; The special existence of chitin fluid has a strong adsorption force to DNA, which can improve the success rate of PCR. At the same time, chitin fluid is composed of many tiny spheres, which can wrap the reaction system, so that the PCR amplification reaction can be completed in a small independent space. , so as to improve the reaction sensitivity of PCR amplification, make the reaction more sufficient, and improve the PCR amplification effect of trace DNA; in addition, chitin fluid can increase the boiling point of the reaction system so that the system buffer is not easy to evaporate, reducing the amount of time in the PCR amplification process. The amount of reagents used, thereby reducing the cost ...
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