Optimization for SRAP-PCR System of Tobacoo Based on Orthogonal Design
Chen Wansheng Wang Yuanying Luo Chenggang Yang Aiguo Jang Caihong Fan Jingyuan Tobacco Research Institute of Chinese Academy of Agricultural Sciences, Qingdao, 266101
In this paper, the orthogonal design was used to optimize SRAP-PCR amplification system on tobacoo (N. tabacum L.) in four levels of five factors (Taq DNA polymerase, Mg2+, DNA template, dNTP, and primer) respectively. The data generated by SRAP-PCR were analyzed by software SPSS V13.0, and the results showed that the effects of each factor in different levels were found and the quantity of Taq DNA polymerase was the most dominant factor affected on the result of PCR. A most suitable SRAP-PCR system for tobacoo was established that is total 25 μL reaction system containing 1.0 U Taq DNA polymerase, 1.5 mmol/L Mg2+, 30.00~120.00 ng DNA template, 0.15 mmol/L dNTP, and 0.40 μmol/L primer. Further, we found out the annealing temperature and cycle number based on the optimized tobacoo SRAP-PCR reaction system, which would be 52℃for SRAP-PCR annealing temperature and 35 cycles for PCR reaction procedure. This optimized system for SRAP marker would become one of the protocols for further research in our lab.