Molecular biology method of early embryo sex determination of transgenic dairy cattle
A technology of molecular biology and genetic modification, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of high semen price, slow separation speed, and semen intolerance to freezing
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[0022] 1. Sample preparation and storage
[0023] Isolate 4-6 cells from bovine embryos to be tested, wash them with sterile ultrapure water for 3 times, put them into a 0.5mL PCR tube, add 10uL of sterile ultrapure water, boil at 100°C for 10min-15min, and immediately place After cooling in crushed ice, centrifuge at 4°C for 5 min, and take the supernatant and store it in a refrigerator at 4°C for later use.
[0024] 2. Primer design
[0025] Y-chromosome-specific primer Forward: 5′-TGATAGTTGATCCCCACAGAAGG-3′
[0026] Reverse: 5′-TGAACTTACAAGCAGCTGAGG-3′
[0027] Autosome-specific primer Forward: 5′-TGAGGCATGGAACTCCGCTTG-3′
[0028] Reverse: 5′-GGTGGTTCCACATTCCGTAGG-3′
[0029] 3. PCR reaction
[0030] Amplify with Y-chromosome-specific primers and autosome internal standard primers. The total volume of the reaction system for PCR amplification is 20uL, of which 10×PCRBuffer 2uL, the concentration of 4dDNTP is 200pmol / L, and th...
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