Optimization of Digoxigenin Based Southern Blot for Transgenic Hevea brasiliensis Analysis

Abstract

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

Li Ji, Lu Xu, Huang Tiandai, Hua Yuwei, Huang Huasun. Optimization of Digoxigenin Based Southern Blot for Transgenic Hevea brasiliensis Analysis[J]. Biotechnology Bulletin, 2014, 0(8): 76-81.

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