Primer, method and kit for quick screening of curly-hair genes of cattle
A kit and gene technology, applied in the field of animal husbandry, can solve the problems of declining milk production and reproductive performance, affecting economic benefits, and stunted growth and development of cattle, so as to reduce the probability of parasitic diseases and improve the economic benefits and costs of breeding. low cost effect
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Embodiment 1
[0032] 1.1 Genomic DNA was extracted from bovine blood by phenol-chloroform extraction, and the DNA sample was diluted to 50 ng / μL for later use. A total of 75 cattle were used in the experiment, including 18 Altay white-headed cattle, 20 Nanyang cattle, 17 Xinjiang brown cattle, and 20 Luxi yellow cattle.
[0033]1.2 According to the gene sequence of KRT27 with Genbank accession number AC_000176.1 (41631641..41637275), use Primer Premier5.0 software to design specific primers for amplifying the bovine KRT27 gene fragment. Exon 1 of the KRT gene is 443bp in total. When designing primers, attention should be paid to the length of the amplified fragment greater than 443bp, the length of the primer is 15-30bp, the GC content is 40%-60%, and the Tm is between 55-80°C, while avoiding the product secondary structure area. In summary, a pair of primers specially designed for KRT gene amplification, the length of the amplified product is 469bp, the length of the primer is 20bp, the G...
Embodiment 2
[0057] 2.1 Genomic DNA was extracted from the blood of 23 Simmental cattle by phenol-chloroform extraction. Utilize the PrimerPremier5.0 software to design specific primers for amplifying the bovine KRT27 gene fragment, and the primer sequences used are:
[0058] The upstream primer sequence is: SEQ ID NO.1:
[0059] 5'-GAGCGTGCGCTTTTCTTCTG-3'
[0060] The downstream primer sequence is: SEQ ID NO.2:
[0061] 5'-TAAACCTCACGGCACTGTGT-3'
[0062] 2.2 According to the above designed primers for detecting bovine curly hair gene carriers, the following steps are used to amplify the bovine KRT27 gene fragment, and the amplified fragment is detected by agarose gel electrophoresis with a mass concentration of 1%.
[0063] (1) Use 25 μl of PCR reaction system: DNA (100ng / μl) 1 μl, upstream and downstream primers (10 μmol / L) 0.5 μl each, 2×Taq PCR MasterMix 12.5 μl, double distilled water (ddH 2 O) Make up to 25 μl.
[0064] (2) PCR reaction conditions: denaturation at 94°C for 4 mi...
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