Biotechnology Bulletin ›› 2014, Vol. 0 ›› Issue (8): 76-81.

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Optimization of Digoxigenin Based Southern Blot for Transgenic Hevea brasiliensis Analysis

Li Ji1, Lu Xu1,2, Huang Tiandai1, Hua Yuwei1, Huang Huasun1   

  1. 1. Rubber Research Institute of Chinese Academy of Tropical Agricultural Sciences & Key Laboratory of Rubber Biology, Ministry of Agriculture, Danzhou 571737;
    2. College of Agriculture, Hainan University, Haikou 570228
  • Revised:2014-03-28 Online:2014-08-15 Published:2014-08-01
  • Contact: 通讯作者: 黄华孙,男,研究员,研究方向:橡胶树种质创新与遗传育种;E-mail:xjshhs@163.com
  • Supported by:

    国家自然科学基金项目(31200503);现代农业产业技术体系建设专项(NYCYTX-34)

Abstract:

Southern blot technology based on the DIG-chemiluminescent detection has been widely used in numerous species. Using Hevea brasiliensis as materials, the DIG-labeled Southern blot analysis was improved through optimizing several key steps, including extraction of genome DNA, usage of DNA samples amount, digestion system, the comparison between PCR and random-primed labeled method and a serial of procedure in the process of the hybrid. The results showed that desirable digestion result could be achieved when at least 40 μg DNA samples with high quality were enzymed in 300 μL reaction system for 12 hours. The efficiency of probe labeled with the method of PCR was higher than that of probe labeled with random primer obviously, while the Southern blot result of PCR labeled was clear, and the background was lightly, which was conducive to read the copy number accurately. Finally, Southern blot with DIG-labeled was optimized, and good result with high sensitivity and low background was achieved.

Key words: Hevea brasiliensis, Transgene, Southern blot, DIG labeling, PCR labeled method