| 摘要: |
| 本研究对模板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，以谱带是否可进行遗传学解释目测比较谱带条数、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25uL和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、0.6U Taq酶、正反向引物各0.2uM；最优反应程序为 94℃预变性2 min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 min。 |
| 关键词: 湿加松 SSR，体系优化 |
| DOI： |
| 分类号: |
| 基金项目:国家自然科学基金,国家重点基础研究发展计划 |
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| SSR-PCR Optimization for Pinus elliottii var. elliottii×P. caribaea var. hondurensis |
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Chen Kao-ke, Huang Shao-wei
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South China Agricultral University
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| Abstract: |
| Template DNA, Mg2+, dNTPs, Taq enzyme, denaturalized temperature, anneal temperature and cycles of SSR-PCR were considered to optimize. The optimized reaction system was consisted of the following components in a 25-µl total volume: 10ng of template DNA, 1×Taq polymerase reaction buffer (including 2.5 mM Mg2+), 0.2 mM of each dNTPs, 0.6 U of Taq DNA polymerase, 0.2uM of former primer DNA and 0.2uM 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℃. |
| Key words: Pinus elliottii var. elliottii×P. caribaea var. hondurensis, SSR, optimization |
