摘要
本试验研究了辣椒SSR-PCR反应体系的主要成分对扩增结果的影响,同时进行了退火温度梯度和循环次数试验。优化后的反应体系为:总体积20μL,1.5μL模板DNA(25ng/μL)、1U Taq酶、0.375μmol.L-1引物、1.875mmol.L-1Mg2+、0.2mmol.L-1dNTPs、1XPCR buffer。扩增程序为:94℃预变性4min,94℃变性45s,58℃(以SSR005为例)退火45s,72℃延伸90s,35个循环,最后一个循环延伸增加为8min,4℃保存至电泳。
The factors which affected the SSR-PCR results of Pepper were studied in the experiment,and the anneal temperature and the cycling were tested. The reaction system optimized was as follows： the 20μL reaction system contained DNA tamplate 1.5μL （25 ng/μL） , Taq DNA polymerase 1U, Primer 0.375μmol.L^-1, Mg^2＋ 1.875mmol.L^-1, dNTPs 0.2mmol.L^-1 and PCR buffer 1 X. And amplification protocol was of pretreatment at 94℃ for 4min , then denaturing at 94℃ for 45 sec, annealing primers at 58℃ for 45 sec（taking SSR005 for example）, extending at 72℃ for 90 sec, cycling 35 times, last time extending for 8 min and then preserving at 4℃ till electrophoresis.
出处
《辣椒杂志》
2007年第2期36-40,共5页
Journal of China Capsicum
基金
北京市基金项目
十五攻关项目(2004BA525B0807)
