摘要
以风信子基因组DNA为ISSR-PCR扩增模板,采用单因素试验方法,对影响PCR扩增体系中Mg2+浓度、dNTPs、模板DNA及引物浓度、Taq酶的用量5个因素进行研究,建立了风信子ISSR-PCR扩增最佳反应体系,即:20μL反应体系中分别加入2μL10×Buffer、1.4μL Mg2+(25mmol/L)、1.5μL dNTPs(2.5mmol/L)、1.5μL引物(10pmol/μL),0.2μLTaq酶(5U/μL),1.2μL模板(30ng/μL),ddH2O补足体积。并以此体系对110条引物进行筛选,最终获得了多态性高,重复性好的引物12条。
Genomic DNA extracted from Hyacinth was used as template for ISSR-PCR, the impact factors in PCR system, Mg2＋ concentration, dNTPs, template DNA, primer concentration and Taq polymerase, were studied using a single factor experiment method by setting up different volume gradient to establish the hyacinth ISSR-PCR reaction system. The experiment results showed that the best reaction system was adding in turn with 2 μL 10×Buffer, 1.4 μL Mg2＋ （25 mmol/L）, 1.5 μL dNTPs （2.5 mmol/L）, 1.5 μL primer （10 pmol/μL）, 0.2 μL Taq polymerase （5 U/μL, 1.2 μL template （30 ng/μL）, and ddH2O to 20 μL at last. According to this PCR system, 110 primers were selected, eventually thirteen primers showed high polymorphism and repeatability.
出处
《分子植物育种》
CAS
CSCD
北大核心
2013年第1期139-144,共6页
Molecular Plant Breeding
基金
中国博士后科学基金面上项目(20100470217)
江苏省高校优势学科建设工程项目共同资助
