河南农业大学学报2012，Vol.46Issue(5)：535-541,7.
泡桐MSAP体系建立及引物筛选
Establishment of Paulownia MSAP reaction systems and primers screening
摘要
Abstract
The establishment of Paulownia MSAP reaction system and its primer screening were investigated through study on the main factors which affected the system quality. The results indicated that the optimum conditions for restriction enzyme digestion of the genomic DNA from the paulownia plant were as follows;300 ng DNA template, 16 U EcoR Ⅰ and Hap Ⅱ (Msp I )respectively in 25 μL reaction volume at 37℃ for 8 h, and inactivated at 80℃ for 20 min; the ligation reaction ( 25 μL ) with 20 μL digestion product ,0.16 μmol · L-1 EcoR I adapter ,1.6 μmol · L-1 Hap Ⅱ ( Msp Ⅰ) adapter ,2.5 μL 10 ×T4 buffer,2 U T4 ligation enzyme at 22 ℃ for 18 h was the optimum condition; the preamplifica-tion reaction (20 μL) with 5 μL the ligation fragment which was diluted 10 times, 100 μmol · L-1 dNTP, 0.5 U Taq enzyme, EcoR Ⅰ and Hap Ⅱ (Msp I) primer respectively 0. 25 μmol · L-1 ,2.5 μL 10 x PCR buffer was the optimum condition; the selective amplification reaction ( 20 μL ) with 5 μX preamplification product which was diluted 30 times, 100 μmol · L dNTP, 0. 5 U Taq enzyme, EcoR I and Hap Ⅱ (Msp Ⅰ ) primer respectively 0. 3 μmol · L-1 ,2. 5 μL 10 x PCR buffer. Finally, by using the optimized system,96 pairs of primers were chosen for the MSAP analysis of paulownia plants.关键词
泡桐/MSAP/反应体系/引物筛选Key words
Paulownia/ MSAP/ reaction system/primer screening分类
农业科技引用本文复制引用
曹喜兵,赵改丽,范国强..泡桐MSAP体系建立及引物筛选[J].河南农业大学学报,2012,46(5):535-541,7.基金项目
国家自然科学基金项目(30571496) （30571496）
