柱花草DNA提取及ISSR反应体系的正交优化

DNA Extraction and Optimization of ISSR-PCR System with Orthogonal Design for Stylosanthes Spp.

报道了以新鲜嫩叶提取高纯度、完整性好的柱花草基因组DNA方法。并利用正交设计,从Taq聚合酶、Mg2+、dNTP、DNA模板、引物浓度5个因素、4个水平对柱花草ISSR反应体系进行优化试验,确立了适合柱花草的快速而又高效的ISSR反应体系,即20μL反应体系中含1×PCR缓冲液,2.0mmol/L Mg2+,0.2mmol/L dNTP,0.4μmol/L引物,60ng DNA模板,1UTaq聚合酶。

A method for isolating highly pure and integrate genomic DNA from fresh young leaves of Stylosanthes spp. was developed, and an orthogonal design was used to optimize the ISSR-PCR system for Stylosanthes DNAs at four levels of five factors（Mg^2＋,dNTP, DNA template,Taq DNA polymerase,and primer）, respectively.As a result,a satisfactory ISSR-PCR system for isolating Stylosanthes DNAs was established,e.g, the 20μL ISSR-PCR system containing 2.0 mmol/L Mg^2＋,0.2 mmol/L dNTP,60 ng DNA template,1 U Taq DNA polymerase,and 0.4 μmol/L primer.