草地学报

›› 2004, Vol. 12 ›› Issue (3): 219-222,230.DOI: 10.11733/j.issn.1007-0435.2004.03.011

• 研究论文 • 上一篇    下一篇

多花胡枝子基因组DNA提取与RAPD反应体系优化

张吉宇1, 袁庆华2, 张文淑2, 鲁挺3   

  1. 1. 兰州大学草地农业科技学院, 甘肃草原生态研究所, 兰州, 730020;
    2. 中国农科院畜牧研究所, 北京, 100094;
    3. 甘肃农业大学草业学院, 兰州, 730070
  • 收稿日期:2003-06-12 修回日期:2003-11-24 出版日期:2004-08-15 发布日期:2004-08-15
  • 通讯作者: 袁庆华,E-mail:yuanqinghua@hotmail.com
  • 作者简介:张吉宇(1978- ),男,甘肃民乐人,硕士,主要从事牧草育种及草地生态研究,E-mail:zjyfrank@163.net
  • 基金资助:
    国家"十五"科技部基础性工作专项资助项目(2001～2003年)

Genomic DNA Extraction and Optimizing RAPD Procedure of Lespedeza floribunda

ZHANG Ji-yu1, YUAN Qing-hua2, ZHANG Wen-shu2, LU Ting3   

  1. 1. College of Pastoral Agriculture Science and Technology of Lanzhou University, Gansu Grassland Ecological Research Institute, Lanzhou, 730020, China;
    2. Institute of Animal Science, CAAS, Beijing, 100094, China;
    3. Grassland College of Gansu Agriculture University, Lanzhou, 730070, China
  • Received:2003-06-12 Revised:2003-11-24 Online:2004-08-15 Published:2004-08-15

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摘要： 以多花胡枝子干种子、萌发种子及开花期叶片三种材料提取基因组DNA,其中开花期叶片用CTAB法提取、干种子和萌发种子用SDS法提取的DNA均用于RAPD研究。结果表明,叶片基因组DNA的RAPD扩增最佳反应体系是:DNA模板用量为75ng,Mg2+浓度2.1mmol/L,dNTP浓度200μmol/L,随机引物浓度为0.2pm/μL,10×buffer2.5uL,TaqDNA聚合酶1.5个单位,反应总体积25uL;扩增反应程序:94℃预变性3min;94℃变性30s,36℃退火30s,72℃延伸1min,循环45次,72℃延伸10min。

关键词: 草原学, 多花胡枝子, DNA提取, RAPD

Abstract: A RAPD analysis was studied with the genomic DNA extraction of Lespedeza floribunda from its leaves at the flowering period,its dried seed,and its germinating seed using the CTAB method and the SDS method.An optimal reaction system and conditions are composed by 75 ng template DNA, 2.1 mmol/L Mg2+, 200 μmol/L dNTP,0.2 pm·μL-1,10×buffer 2.5 uL, 1.5 U TaqDNA polymerase,25 uL total reaction buffer. Thermal reaction program suitable for the RAPD analysis was devised as followings: an initial denaturation 94℃ for 3 minutes;45 cycles of 94℃ for 30 seconds,36℃ for 30 seconds,72℃ for 1 minute;and a final extention at 72℃ for 10 minutes.

Key words: Grassland science, Lespedeza floribunda, DNA extraction, RAPD

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