Duplex PCR microsatellite primers for paternity testing of procambarus clarkii and application
A technology of Procarydon clarkii and paternity testing, which is applied in the field of animal molecular genetics to avoid inbreeding, great practical value, and reduction of financial and human resources.
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Embodiment 1
[0019] Extraction of Procambarus clarkii DNA:
[0020] 1. Take the Procambarus clarkii, collect the tail muscles of the Procambarus clarkii, and extract the genomic DNA of the Procambarus clarkii. The extraction method adopts the traditional phenol-chloroform extraction method.
[0021] 1.1 Take 0.1g of caudal fin from each individual and put it in a 1.5ml centrifuge tube, cut it into pieces, add 450μL STE extraction buffer (10mmol / L Tris-HCl, pH8.0; 1mmol / L EDTA, pH8.0), 35μL SDS ( 10%), 15 μL proteinase K (0.2%).
[0022] 1.2 Place the centrifuge tube in a water bath at 55°C for 1 hour until it becomes clear and transparent.
[0023] 1.3 Add 700ul Tris saturated phenol into the centrifuge tube, mix on a shaker for 30 minutes, centrifuge at 12,000 rpm for 10 minutes at 4°C, and transfer the supernatant into another clean eppendorf tube (note that the 1mL tube cut flat with the tip Pipette the supernatant to avoid confusing the lower layer).
[0024] 1.4 Add an equal volume...
Embodiment 2
[0029] Test the allelic band size for each primer:
[0030] Purchase 24 individual mature Procambarus clarkii from various places in Wuxi, Hangzhou, Qianjiang, and Jinan, respectively extract the DNA of these 96 individuals, and use the primers described in Table 1 to test the presence of allelic bands in these 96 individuals. Size, each primer forms a PCR reaction system, the reaction system is: 10×PCR Buffer 2.5ul, 2.5mmol / L dNTP 0.5ul, MgCl21.5ul, each set of reaction system upstream and downstream primers 1ul, Taq enzyme 0.3ul, DNA Template 3ul, ultrapure water 15.2ul. The PCR reaction program was: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing temperature for 30 s, extension at 72°C for 30 s, 30 cycles; extension at 72°C for 10 min; storage at 4°C. PCR products were electrophoresed and silver stained with 10% polyacrylamide gel. Use the software BIO-PROFIL to judge the size of the allele fragments of each individual displayed on the polyacr...
Embodiment 3
[0035] Randomly get 24 Procambarus clarkii, extract DNA, test the alleles in these 24 individuals with the primers described in claim 1, each group forms a PCR reaction system, and the reaction system is: 10 × PCR Buffer 2.5 ul, 0.5ul of 2.5mmol / LdNTP, 1.5ul of MgCl2, 1ul of upstream and downstream primers of two pairs of primers in each reaction system, 0.3ul of Taq enzyme, 3ul of DNA template, and 13.2ul of ultrapure water. The PCR reaction program was: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing temperature for 30 s, extension at 72°C for 30 s, 30 cycles; extension at 72°C for 10 min; storage at 4°C. PCR products were electrophoresed and silver stained with 10% polyacrylamide gel. Use the software BIO-PROFIL to judge the size of the allelic fragments of each individual displayed on the polyacrylamide gel to see whether there is a chain reaction or dimer between the primers. The results are as follows: figure 2 shown. The results showed th...
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