红锥基因组RAPD反应体系的建立和优化

The Optimization of RAPD Reaction System for Castanopsis hystrix A. DC.

摘要:

以红锥（Castanopsis hystrix A. DC.）嫩叶为材料，应用改良的CTAB方法成功提取了红锥基因组DNA，并对影响随机扩增多态DNA（RAPD）反应的各因素进行了优化，建立了红锥RAPD的优化反应体系及程序。在25μl反应体积中，各组分浓度为：模板DNA 0.8 ng μl-1, 10×Buffer 2.5μl, Mg2+ 2.0 mmol/L, Taq DNA 聚合酶0. 8 U, dNTPs 0.35 mmol/L, 随机引物S42 0.28 μmol/L。PCR循环程序为：94℃预变性3 min, 然后94℃ 30 s，39℃ 1 min, 72℃ 2 min, 35个循环，最后72℃延伸10 min, 4℃保存。

Abstract:

The genomic DNA was extracted from Castanopsis hystrix A. DC. using the modified method of CTAB, and the optimization of its RAPD reaction system was also studied. Results showed that the high-quality genomic DNA can be obtained with the modified method of CTAB, and it could be directly used for RAPD analyses. The optimal PCR system for RAPD analysis was as follows: 0.8 ng μl-1 DNA template, 2.0 mmol/L Mg2+, 0.8 U Taq polymerase, 0.35 mmol/L dNTPs, 0.28 μmol/L random primer, in 25 μl reaction system. RAPD program is 3 minutes at 94℃ for predenaturation, then followed by 35 cycles, each with 30 seconds at 94℃ for denaturation, 1minute at 39℃ for annealing , 2 minutes at 72℃ for extension, finally extension at 72℃ for 10 minutes.