南京林业大学学报（自然科学版）2012，Vol.36Issue(5)：42-46,5.
东方百合ISSR-PCR反应体系的正交优化
Optimizing the ISSR-PCR reaction system for oriental lily based on orthogonal design
摘要
Abstract
'Constanta' and ' Sorbonne' and 9 F1 hybrids of ' Constanta' × ' Sorbonne' were used as materials in this experiment, and a modified method of CTAB was applied to extract the genomic DNA from the young leaves. The orthogonal design L16 (45 ) was used to optimize the ISSR - PCR system for oriental lily at four levels and five factors, namely, DNA polymerase, Mg2+ , DNA template, dNTPs and primers. The results of PCR were evaluated by intuitive and variance analysis. Results showed that the best reaction system was 20 μL reaction solution, containing 0. 5 μmol/min Taq DNA polymerase, 1.75 mmol/mL Mg2+ , 40 - 100 ng template DNA, 0. 15 mmol/mL dNTPs, 0. 6 μmol/L primers, 2 μL 10 × PCR buffer and ddH2O. Using single factor experiment design, we found that 55-45 ℃. was the best touch down PCR annealing temperature, the best cycle times was 30 and the best extend time was 1. 5 min. The stabilization of the optimized reaction system was tested by using ' Constanta' and ' Sorbonne' and 9 F1 hybrids of ' Constanta' × ' Sorbonne' .关键词
东方百合/ISSR-PCR/正交设计Key words
oriental lily/ ISSR - PCR/ orthogonal design分类
农业科技引用本文复制引用
丁信誉,邱帅,席梦利,施季森..东方百合ISSR-PCR反应体系的正交优化[J].南京林业大学学报（自然科学版）,2012,36(5):42-46,5.基金项目
国家自然科学基金项目(30972407) （30972407）
江苏高校优势学科建设工程资助项目(PAPD) （PAPD）
