Agarose gel electrophoretic separation of the 5′RACE and overlapping 3′RACE products of zebrafish wdr81 transcript. a Expected size of the 5′RACE amplicon based on the experimental design was obtained and one amplicon was observed as a PCR product indicating that there is one 5′ UTR structure of zebrafish wdr81. The band of ~692 bp was excised, cloned and sequenced. Lane 1 5′RACE product of 24 hpf embryo wdr81 cDNA; lane 2 5′RACE product of brain wdr81 cDNA; lane 3 negative control without template, M pUC mix marker 8 (SM0301, Thermo Scientific). Expected size of the overlapping 3′RACE amplicons based on the experiment design were obtained from the PCR experiments with primer pairs 1 (data not shown) and 3 from the Table 3. The resulting amplicon obtained with primer pair 2 was longer than the expected amplicon size indicating presence of an insertion site. One amplicon per reaction was obtained as PCR products concluding that there is one transcript of zebrafish wdr81.
b Amplification with primer pair 3 from Table 3. c Amplification with primer pair 2 from Table 3. In both gels, b and c, lane 1 24 hpf embryo RACE ready cDNA, lane 2 brain RACE ready cDNA, lane 3 no template negative control. M MassRuler Mix DNA Marker (SM0403, Thermo Scientific)