中国沙漠 ›› 2006, Vol. 26 ›› Issue (2): 286-290.

• 研究简报 • 上一篇    下一篇


郝瑞文1, 景建洲2, 李振勇2, 贾敬芬1*   

  1. 1.西北大学 生命科学学院, 陕西 西安 710069; 2.华美生物工程公司博士后工作站, 河南 洛阳 471003
  • 收稿日期:2004-11-22 修回日期:2005-01-24 出版日期:2006-03-20 发布日期:2006-03-20

Optimization of RAPD Analytic Conditions on Genomic DNA of Sarcozygium xanthoxylon

HAO Rui-wen1, JING Jian-zhou2, LI Zhen-yong2, JIA Jing-fen1   

  1. 1.College of Life Sciences, Northwest University, Xian 710069, China; 2.Postdoctoral Programme, Sino-American Biotechnology Company, Luoyang 471003, Henan, China
  • Received:2004-11-22 Revised:2005-01-24 Online:2006-03-20 Published:2006-03-20

摘要: 实验以霸王20 d龄无菌籽苗为材料,使用CTAB法提取其基因组DNA,进行RAPD条件优化分析。通过单因子实验分别研究了Mg2+浓度、dNTPS浓度、Taq酶的浓度、引物浓度和模板DNA浓度对RAPD反应的影响,即在25 μL总反应体系中,Mg2+的适宜浓度为1.5~2.5 mmol\5L-1、dNTPs的适宜浓度为0.2~0.3 mmol\5L-1、随机引物的浓度以1.2 μmol\5L-1为宜、Taq酶的用量以2.0 U为佳、模板DNA的最适用量为50 ng。本研究的PCR扩增程序为94℃预变性8 min, 94℃变性1 min, 37℃退火1 min, 72℃延伸2 min,设40个循环,最后72℃保温6 min。

关键词: 霸王, RAPD, PCR, 条件优化

Abstract: Sarcozygium xanthoxylon Maxim genomic DNA, extracted from its sterile 20 days old seedlings by CTAB method, was used as RAPD amplification template. The effects of main elements, including concentration of Mg2+, dNTPs, DNA, primer and Taq polymerase dosage on RAPD amplification were studied through single factor experiment. A following efficient protocol for optimal reaction system of RAPD analysis in Sarcozygium xanthoxylon Maxim was established , i.e , the 25 μL reaction mixture contained 1.5~2.5 mmol·L-1 Mg2+, 0.2~0.3 mmol·L-1 of each dNTPs, 1.2 μmol·L-1 arbitrary primer, 2.0 U Taq polymerase, 50 ng template DNA . The amplification was performed as follows: 94℃ pre-denaturation for 8 min, 40 cycles of 94℃ for 1 min, 37℃ for 1 min, 72℃ for 2 min, and 72℃ extension for 6 min.

Key words: Sarcozygium xanthoxylon Maxim, RAPD(Random amplified polymorphic DNA), PCR(Polymerase chain reaction), optimization