Establishment and optimization of SRAP-PCR system in Canavalia ensiformis
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摘要:
【目的】建立和优化剑豆 Canavalia ensiformis 的SRAP-PCR体系,为分析剑豆遗传多样性和建立遗传图谱打下基础.【方法】首先用单因素试验法对SRAP-PCR反应体系的5个主要影响因素(Mg2+、dNTPs、引物、Taq DNA聚合酶、模板DNA)各设8个浓度梯度进行试验,找到5个因素的适宜浓度范围,再采用均匀设计法进行5因素4水平和5因素3水平的2轮优化.【结果和结论】建立了剑豆SRAP-PCR最佳反应体系(25 μL):Mg2+ 1.75 mmol/L,dNTPs 200 μmol/L,引物0.36 μmol/L,Taq DNA 聚合酶0.06 U/μL,模板 DNA 40 ng.所建立的体系稳定可靠,适用于剑豆后续的SRAP分析.
Abstract:
【Objective】The purpose of this study is to establish the SRAP-PCR reaction system for Canavalia ensiformis. 【Method】An optimization experiment with single factor design was conducted, comprising five factors of Taq DNA polymerase, Mg2+, primer, dNTPs and DNA template, each with eight concentration levels,aiming to screen their suitable concentration range. After that, uniform design U16(45)and U12(35) were operated in order to improve the accuracy. 【Result and conclusion】The results showed that the optimum SRAP-PCR system was established, including Mg22+ 1.75 mmol/L, dNTPs 200 μmol/L,primers 0.36 μmol/L, Taq DNA polymerase 0.06 U/μL and DNA template 40 ng in the 25 μL reaction system. The reaction system is steady and dependable, which can be applied to the analysis of Canavalia ensiformis by SRAP.