芹菜RAPD反应体系的优化研究

本试验以一般RAPD反应程序为基础，采用单因素递进筛选方法，针对Taq DNA聚合酶、Mg2+、dNTPs、随机引物和DNA模板5个主要影响因素，分别设置5个不同浓度梯度，对芹菜进行RAPD-PCR扩增，建立了芹菜RAPD技术最优体系。结果表明：25 μL反应体系中含Taq DNA聚合酶1.0 U、Mg2+ 3.0 mM、dNTPs 0.2 mM、引物28 ng、模板DNA 70 ng，10×PCR Buffer 2.5 μL；扩增程序为：94℃预变性5 min，94℃变性1 min，36℃退火1 min，72℃延伸1 min，进行42个循环，最后72℃延伸10 min。

The Optimization of RAPD Reaction System in Celery

In the present study,we set up separately five different concentration gradient to five main influence factors （dNTPs,Taq DNA polymerase,Mg 2＋,random primer and DNA template）.Then based on the common RAPD reaction system,using the method of single factor progressive screening,we conducted the RAPD-PCR amplification of celery.Finally,we got the optimization of RAPD reaction system in celery.Conclusion of the optimized contents of 25 μL RAPD reaction system was as follows：Taq DNA polymerase 1.0 U,Mg 2＋ 3.0 mM,dNTPs 0.2 mM,primer 28 ng,DNA templet 70 ng,10×reaction Buffer 2.5 μL.The optimized PCR cycle program was as follows：94℃ for 5 min,94℃ for 1 min,36℃ for 1 min,72℃ for 1 min,42 cycles and at final 72℃ for 10 min.