STMN2 gene as sheep immune trait molecular marker and use thereof
A molecular marker, sheep technology, applied in recombinant DNA technology, microbial determination/inspection, DNA/RNA fragments, etc., can solve problems affecting the plasticity of the nervous system, etc.
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Embodiment 1
[0029] Cloning of embodiment 1, STMN2 gene
[0030] (1) Primer sequence design
[0031] According to the sheep STMN2 gene sequence (GenBank accession number: XM_004011755.2), a pair of primers M‐F and M‐R were designed using Primer5.0 software, and the specific sequences are as follows:
[0032] STMN2: M-F: 5'-CCTGCCTCTTGCTCTTTCTC-3', (ie: the sequence shown in SEQ ID NO: 3);
[0033] M-R: 5'-TGTCATACATTTCCCATACTG-3'; (ie: the sequence shown in SEQ ID NO: 4).
[0034] (2) Cloning and sequencing of PCR products
[0035] Ligate the purified PCR product with the pMD‐18T vector (purchased from Treasure Bioengineering Dalian Co., Ltd.) in a water bath at 4°C overnight; take 100-120 μL of competent cells in a 1.5 mL Pendorff tube under sterile conditions, and put 5 μL of the ligated product Add and mix well, place on ice for 30 min, heat shock at 42°C for 90 s, then ice-bath for 3-4 min, add 400 μL LB liquid medium without antibiotics, and incubate with shaking at 37°C for 45 min...
Embodiment 2
[0038] Embodiment 2, establishment of PCR-RFLP diagnostic method
[0039] (1), primer sequence design
[0040] STMN2: M-F: 5'-GTTGTTCAGCCAGGTTTGTG-3', (ie: the sequence shown in SEQ ID NO: 5);
[0041] M-R: 5'-ACCAGCCTCCCCTCAGTCTAT-3'; (ie: the sequence shown in SEQ ID NO: 6).
[0042] (2) PCR amplification conditions
[0043] The total volume of the PCR reaction is 20 μL, including about 100 ng of sheep genomic DNA, containing 1 times the buffer (purchased from Promega Company), 1.5 mmol / LMgCl 2 , the final concentration of dNTP is 150 μmol / L, the final concentration of primer is 0.4 μmol / L, 2UTaq DNA polymerase (Promega). The PCR amplification program was: 94°C for 3min, 35 cycles of 94°C for 30s, 65°C for 30s, then 72°C for 25s, and finally 72°C for 5min. PCR reaction products were detected by 2% agarose gel electrophoresis. A 476bp specific amplified fragment was obtained, which was located at the 4th exon (such as figure 2 ). As a result of the sequencing, it was ...
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