巨竹ISSR反应体系的建立与优化

以巨竹叶片提取的基因组DNA为材料,用引物UBC810(序列为GAG AGA GAG AGA GAG AT)研究了PCR反应体系的主要成分、退火温度及循环次数对该种植物ISSR扩增结果的影响。结果表明,20μL的反应体系含40 ng模板DNA、0.6μmol.L-1引物,1.0 U Taq DNA聚合酶,2.5 mmol.L-1Mg2+,0.25 mmol.L-1dNTPs,1×Buffer。PCR扩增程序为:94℃预变性5 min;94℃变性45 s,54.5℃复性30 s,70℃延伸90 s,循环40次;72℃延伸10 min,置4℃保存。

Establishment and optimization of ISSR amplification system in Gigantochloa levis

Taking genomic DNA extracted from Gigantochloa levis leaves as the template material,the concentrations of PCR components,annealing temperature and cycles,which affected the ISSR amplification,were optimized with the primer UBC810(primer sequence:GAG AGA GAG AGA GAG AT).The results showed that the 20 μL ISSR reaction system included 40 ng template DNA,0.6 μmol·L-1 primer,1.0 U Taq DNA polymerase,2.5 mmol·L-1 Mg2+,0.25 mmol·L-1 dNTPs,1×Buffer.The optimal PCR amplification process was 5 minutes at 94 ℃ for predenaturation,followed by 40 cycles,each with 45 seconds at 94 ℃ for denaturation,30 seconds at 54.5 ℃ for annealing,90 seconds at 72 ℃ for extension,finally extension at 72 ℃ for 10 minutes and holding the samples at 4 ℃.