Dig 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).

Oligonucleotide probes containing microsatellite sequences (CA)₁₅, (CAA)₁₀, (CAC)₁₀, (CAG)₁₀, (CAT)₁₀, (CGG)₁₀, (GA)₁₅, (GAA)₁₀ and (TA)₁₅ were directly labeled with Cy5 during synthesis by Sigma (St. Louis, MO, USA), as described by Kubat and colleagues [22] (link). The retrotransposable elements Rex 1, 3 and 6 were obtained by polymerase chain reaction (PCR) [23] (link). The 18S rDNA probe, corresponding to a 1,400-bp segment of the 18S rRNA gene, was obtained via PCR from the nuclear DNA according to Cioffi and colleagues [24] (link). All probes were labeled with DIG-11-dUTP using DIG-Nick-translation Mix (Roche, Mannheim, Germany) and were used for the fluorescence in situ hybridization (FISH) experiments.

Fluorescence in situ hybridization (FISH) experiments were performed with a standard protocol. The pPur (Clontech, 631601) plasmid was labeled with FITC fluorescent dye and used as a probe for ACE detection. We used the Roche DIG Nick Translation Mix (1745816) or the Roche Biotin Nick Translation Mix (1745824) for labeling DNA probes.

The GTG- and CBG-banding of C. hoffmanni chromosomes were performed according to Seabright^{67 (link)} and Sumner^{68 (link)}, respectively. FISH was performed using the cloned satDNA sequences as probes after they were labeled by nick-translation with digoxigenin-11-dUTP (DIG-Nick Translation mix, Roche Applied Science) for SATCHO1 and biotin-16-dUTP (Biotin-Nick Translation mix, Roche Applied Science) for SATCHO2. The probes (~ 150 ng in 50% formamide/2xSSC) were denatured for 10 min at 98 °C. Chromosomes were dehydrated in ethanol series (70%, 90%, 100%) and denatured in 70% formamide/2xSSC for 2 min at 75 °C. The hybridization was performed overnight at 37 °C. Post-hybridization washes comprised two 5 min incubations in 2xSSC at 45 °C. Immunodetection was performed with anti-digoxigenin conjugated with rhodamine (Roche Applied Science) for SATCHO1 and avidin conjugated with FITC (Roche Applied Science) for SATCHO2. Chromosomes were counterstained with DAPI 1:500 in Slowfade (Invitrogen). The analysis was performed under a Zeiss Axioimager 2 epifluorescence microscope adapted with a CCD camera and image acquisition was performed with the AxioVision (Zeiss) software (Carl Zeiss MicroImaging, Jena, Germany).

Larval neuroblast chromosomes from Oregon R were prepared as described previously58 (link). Chromosomes were counterstained with 4′,6-diamino-2-phenylindole (DAPI). The dodeca satellite oligo probe 5′-CCCGTACTGGTCCCGTACTGGTCCCGTACTCGGTCCCGTACTCGGT-3′ and the 10 bp satellite oligo probe 5′-AATAACATAGAATAACATAGAATAACATAGAATAACATAGAATAACATAG-3′ were chemically synthesized and labeled at the 5′ end with Cy3 or at the 3′ end with fluorescein (New England Biolabs). DNA probes derived from clones or PCR products were labeled by nick translation with digoxygenin-11-dUTP (Roche) using the DIG-Nick Translation Mix (Roche). Digoxygenin labeled probes were detected with Anti-Digoxigenin-Rhodamine, Fab fragments (Roche) in a 1:200 dilution, following supplier recommendations. Digital images were obtained using a Zeiss Axiover 200 microscope equipped with a cooled Charge-Coupled Device camera. The fluorescent signals were recorded separately as grey-scale digital images and then pseudo-colored and merged using Adobe Photoshop software.

For probe preparation, a mixture of the 2-kb ApaI fragment of the 3′-untranslated region and the 23Zn-1 fragment containing ORF1+ORF2 of HeT-A39 (link)40 (link) was labelled with digoxigenin-11-dUTP (DIG-Nick Translation Mix, Roche) following the manufacturer's instructions. Three micrograms of sonicated salmon sperm DNA and 80 ng of labelled probe per slide were ethanol precipitated and resuspended in 50% formamide, 10% dextran sulfate and 2 × SSC. Chromosome preparations were dehydrated by sequential immersion in 70%, 90% and absolute ethanol, and then denaturated 2 min at 70 °C in 70% formamide and 2 × SSC. After sequential immersion in cold 70%, 90% and absolute ethanol, slides were incubated with 10 μl of probe at 37 °C in wet chamber. After overnight incubation, slides were whashed three times at 42 °C with 50% formamide and 2 × SSC, followed by three whashes at 60 °C in 0.1 × SSC. Chromosomes preparations were blocked 30 min at 37 °C in 3% BSA, 0.1% Tween 20 and 4 × SSC, and then incubated with 1:50 anti-digoxigenin (Roche 11207750910) dilution in 0.1% BSA, 0.1% Tween-20 and 4 × SSC for 30 min at 37 °C. After three washes at 42 °C in 4 × SSC and 0.1% Tween-20, slides were air dried, mounted in Vectashield medium H-1200 with DAPI and analysed using a Zeiss Axioplan epifluorescence microscope equipped with a cooled CCD camera (Photometrics).