Multiplex PCR (polymerase chain reaction) primers and method for identifying Panthera pardus, and application thereof in identifying Panthera pardus
A golden leopard, multiple technology, applied in the fields of biochemical equipment and methods, recombinant DNA technology, microbial determination/inspection, etc., can solve the problems of time-consuming and labor-intensive, and achieve the reduction of sampling difficulty, economical and practical identification, and simplicity. The effect of identification
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Embodiment 1
[0036] Put 0.5 g of the sample to be tested numbered 5 in Table 1 into a centrifuge tube and cut the sample to be tested in the centrifuge tube with sterilized scissors, then add 500 μL of lysate and 30 μL of 10% ten Sodium dialkyl sulfate and 3 μL of 20 mg / ml proteinase K were thoroughly mixed and then digested in a water bath at 56°C for 12 hours until the liquid in the tube was clear. Add 500 μL of Tris-balanced phenol to the above-mentioned digested mixture, shake slightly for 5 minutes, then place it in a centrifuge at 11000 r / min for 10 minutes, and take the supernatant after centrifugation. Repeat the above centrifugation step twice. Add 1,000 μL of frozen absolute ethanol to the supernatant obtained after repeated centrifugation, place it at -20°C for 1 hour, then centrifuge it in a centrifuge at 12,000 r / min for 13 minutes, discard the supernatant, and add 800 μL of 70% ethanol was shaken slightly for 0.5 min, then placed in a centrifuge at 13000 r / min for 13 min, an...
Embodiment 2
[0039] Operate according to the method of Example 1, the difference is that the sample number to be tested is 12 (in Table 1), the consumption of each primer is 0.75 μ L, the consumption of the total DNA is 45 ng, and the annealing temperature is 55°C, the annealing time is 40s, the electrophoresis results are as follows figure 1 Shown (lane number is 12).
Embodiment 3
[0041] According to the method of Example 1, the amount of each primer used was 1.5 μL, the amount of the total DNA used was 55 ng, the annealing temperature was 60° C., and the annealing time was 25 s. The electrophoresis results were consistent with those in Example 1. same.
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