Bio nick translation mix

The gDNA of H. odoe was used for comparative analyzes with the gDNAs of several Erythrinidae species, namely E. erythrinus (karyomorph D), Hoplias lacerdae, H. malabaricus (karyomorph A) and Hoplerythrinus unitaeniatus (karyomorph D). The gDNA of H. odoe was labeled with biotin-16-dUTP using BIO-nick-translation Mix (Roche), while the male-derived gDNAs of E. erythrinus, H. malabaricus, H. unitaeniatus, and H. lacerdae were labeled with digoxigenin-11-dUTP using DIG-nick-translation Mix (Roche, Manheim, Germany). In all experiments it was utilized C₀t-1 DNA (i.e., fraction of genomic DNA enriched for highly and moderately repetitive sequences), prepared according to Zwick et al. (1997) (link), for blocking common genomic repetitive sequences. The final probe was composed of 500 ng of H. odoe gDNA plus 500 ng of the corresponding gDNA for each Erythrinidae species. The probe was ethanol-precipitated and the dry pellet dissolved in a hybridization buffer (20 μL per slide) containing 50% formamide + 2x SSC + 10% SDS+ 10% dextran sulfate and Denhardt’s buffer, pH 7.0).

The 18S rDNA and (TTAGGG)_n probes were isolated according to Gross et al. [48 (link)] and Ijdo et al. [49 (link)], respectively. Both probes were labeled with digoxigenin-11-dUTP using Dig-Nick kit (Bio-NickTranslation Mix, Roche, Mannheim, Germany) following the manufacturer’s recommendations (Roche, Mannheim, Germany). Microsatellites motifs (AC)₁₅, (AG)₁₅, (ATTC)₈, (ATCC)₈, and (GATA)₈ were used directly labeled with Cy-3 during the synthesis [50 (link)].