OPTIMUM CONDITIONS FOR DGGE OF 16S rDNA FROM SUT TILAPIA INTESTINAL TRACT MICRO FLORA
Keywords:
GC-clamp, 16S rDNA-DGGE, DGGE conditionAbstract
Denaturing Gradient Gel Electrophoresis (DGGE) is a molecular fingerprinting method that separatessimilar size (bp), but different sequence of PCR products using acrylamide gel that contain denaturinggradient of urea and formamide. GC clamp is added at the 5' end of one of the primer to prevent thePCR amplicon from completely denature. Different bacteria have different DNA sequences that willdenature at different denaturant concentrations. Different DGGE conditions are suitable for differentbacterial communities. This research aimed to optimize the conditions for determining bacterialcommunity of fish intestinal tract. The percent of polyacrylamide gel (8.0% and 10.0%), denaturantconcentrations (45 - 55%, 30 - 60%, and 30 - 70%), running times (5 h and 12 h) and voltages used (120 Vand 200 V) were varied. The results indicated that, 8.0% polyacrylamide gel, 30 - 60% denaturant, 12 hrunning time and 120 V was the optimum condition for separation of different species of fish intestinaltract micro flora.
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