大豆RAPD影响因素的探讨

STUDY OF AFFECTING FACTORS IN SOYBEAN RAPD

为确保ＲＡＰＤ结果的重复性和真实性，本文对影响大豆ＲＡＰＤ结果的主要因素进行了研究。这些因素包括：Ｔａｑ酶、ＭｇＣｌ２、模板、ｄＮＴＰｓ、引物浓度，双引物扩增及热循环数。最终优化的大豆ＲＡＰＤ反应体系为：２５ｕｌ反应液中，１０倍反应缓冲液（１００ｍＭＴｒｉｓ－ＨＣｌｐＨ８．０，５００ｍＭＫＣｌ，０．１％ｇｅｌａｔｉｎ），２．０ｍＭＭｇＣｌ２，ｄＣＴＰ、ｄＧＴＰ、ｄＴＴＰ、ｄＡＴＰ各为０．２ｍＭ，１５ｎｇ引物，２５ｎｇＤＮＡ，１ｕＴａｑ酶。研究结果还表明，双引物比单引物扩增的小片段数增多，但也存在着原有单引物扩增条带缺失现象。

To ensure that the results of RAPD will be reproducible and true, the major affecting factors in soybean RAPD were studied. These factors included: the concentrations of Taq polymerase enzyme, MgCl 2, template, dNTPs, primer, amplification of paired primers and thermal cycles. The final optimized soybean RAPD conditions were: in 25ul reaction solution, 10×Buffer (100mM Tris-HCl pH8.0,500mM KCl, 0.1%gelatin), 2.0mM MgCl 2, 0.2mM dNTPs, 15ng primer, 1.0u Taq polymerase enzyme. The results also showed that compared with the amplication of single primer, paired primers not only resulted in more small fragments but also lost some bands.