湿加松的SSR反应体系优化

SSR-PCR Optimization for Pinus elliottii var.elliottii×P.caribaea var.hondurensis

对湿加松SSR反应体系中模板DNA的用量、Mg2+浓度、dNTPs及Taq酶的用量、变性温度、退火温度、反应循环次数共7个影响SSR扩增效果的因素,逐一设置单因素多水平进行试验,以目测比较确定谱带条数、亮度、清晰度、背景透明度以及结果的重复性等为判定反应条件适合程度的指标,其反应体系为25μL,扩增程序经优化后确定为:10 ng的模板、1×PCR缓冲液、2.5 mM的Mg2+、0.2 mM dNTPs、0.6 UTaq酶、正反向引物各0.2μM;最优反应程序为94℃预变性2 m in之后进入循环,每个循环94℃变性45 s,退火45 s,72℃延伸45 s。退火温度从Tm开始,每隔2个循环降低1℃,直至(Tm-10℃),维持40个循环。循环结束,72℃延伸5 m in。

Template DNA, Mg^2＋ , dNTPs, Taq and cycles of SSR-PCR on Pinus elliottii var. elliottii × P. enzyme, denaturalized temperature, anneal temperature caribaea var. hondurensis were considered to optimize. The optimized reaction system was consisted of the following components in a 25 uL total volume： 10 ng of template DNA, 1 × Taq polymerase reaction buffer （ including 2.5 mM Mg^2＋ ）, 0.2 mM of each dNTPs, 0.6 U of Taq DNA polymerase, 0.2 uM of former primer DNA and 0.2 uM of reverse primer DNA. The optimized reaction program was 2 minutes at 94℃, 45 seconds at 94℃, 45 seconds at the starting temperature, 45 seconds at 72℃, followed by a touchdown, drop 1 degree each time, doing 2 cycles at each temperature, to 10 degrees below the starting temperature, follow this with 20 cycles at the lowest temperature, and final extension of 5 minutes at 72℃.