The nested PCR amplified products were analyzed for sequence polymorphism on 40%–60% linear DNA denaturing gradient polyacrylamide gel, 0.8 g·L−1 alongside species-specific markers using DCode system (Bio-Rad, Hercules, CA, USA) for 16 h at 58 °C and 60 V in 1× Tris-acetate-EDTA buffer, pH 8.5.19 (link),30 (link) The gels were stained with ethidium bromide solution (0.5 µg·mL−1) for 15 min and the images digitally captured using Alpha Imager 3300 system (Alpha Innotech San Leandro, CA, USA).
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Nested PCR Sequence Polymorphism Analysis
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Other organizations : New York University, Nyack College
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Variable analysis
independent variables
- DNA denaturing gradient polyacrylamide gel concentration (40%-60% linear)
- Sequence polymorphism of the nested PCR amplified products
- Temperature (58 °C)
- Voltage (60 V)
- Buffer (1× Tris-acetate-EDTA, pH 8.5)
- Run time (16 h)
- Ethidium bromide staining concentration (0.5 µg·mL^-1)
- Imaging system (Alpha Imager 3300 system)
- Species-specific markers
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