Hemibarbus maculates microsatellite sites and primers
A technology of microsatellite sites and fish bones is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc. Achieve high polymorphism and stability
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[0021] 1. Screening of microsatellite loci
[0022] 1. Genomic DNA extraction, digestion and recovery:
[0023] Use phenol extraction to extract genomic DNA from flower fish bones: each individual weighs 100mg of muscle, washes it twice with sterile water, cuts it into pieces and adds 10mM Tris-Hcl pH 8.0, 20mM EDTA pH 8.0, 10mM NaCl, 1% SDS, 600 μl of lysate of 100 μg / ml proteinase K, after digesting the muscle at 55 ° C, centrifuge at 12000 rpm for 10 min, take the supernatant into a new 1.5 ml centrifuge tube, add phenol, chloroform and isoamyl alcohol at a ratio of 25:24:1 And extract twice, the ratio is 24:1 chloroform and isoamyl alcohol After extraction once, the water phase is transferred to a new tube, add -20 ℃ absolute ethanol, centrifuge at 12,000rpm for 10min, wash the precipitate twice with 75% ethanol , dried at room temperature and dissolved in 100 μl 0.1×TE.
[0024] 2. Digestion, recovery and linker connection:
[0025] Take 100 μg of genomic DNA, and part...
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