Colonies containing recombinant bacmids were identified by disruption of the lacZa gene, and the white colonies were chosen for recombinant bacmid DNA isolation. DNA was isolated using a Qiagen plasmid isolation kit (Qiagen Inc.) specific for DNA over 135 kb long. The recombinant bacmid DNAs were analyzed on 0.5% agarose gel and followed by PCR analysis using pUC/M13 forward and reverse primers (Table 1) to confirm the presence as well as the correct insert size of the transposed grouper Ihb and cga cDNA within the bacmid.
To confirm the authenticity of the bacmid, Southern blot analysis was conducted. After capturing the image of the agarose gel electrophoresis, the bacmids were transferred by vacuum and cross-linked to Hybond-N nylon paper (B.M.) following the instruction manual. After overnight hybridization at 42°C the blot was washed with 2XSSC, 0.1% SDS for 5 min twice at room temperature and then with 0.5XSSC, 0.1% SDS for 15 min twice at 65 °C. After the membrane was submerged into the detection buffer with CDP-Star substrate, the chemiluminescent signals from the membrane were detected with the GeneGenome System (SynGene, UK).