剑豆SRAP-PCR反应体系的建立及优化

刘明骞; 陈丽君; 欧阳昆唏; 丁美美; 陈晓阳

doi:10.7671/j.issn.1001-411X.2015.01.014

剑豆SRAP-PCR反应体系的建立及优化

Establishment and optimization of SRAP-PCR system in Canavalia ensiformis

摘要

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(Mg^{2 +}、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 Mg²⁺ 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.

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