Dako telomere pna fish kit fitc

Two tandemly arrayed rDNA probes were obtained by PCR from the nuclear DNA of Harttia intermontana. The 5S rDNA probe included 120 base pairs (bp) of the 5S rRNA transcript region and 200 bp of a non-transcribed spacer (NTS), isolated according to Pendás et al. [29 (link)]. The 18S rDNA probe contained a 1400 bp segment of the 18S rRNA gene and was isolated following Cioffi et al. [30 (link)]. The probes were directly labeled with the Nick-Translation mix kit (Jena Bioscience, Jena, Germany) using ATTO550-dUTP for the 5S rDNA and AF488-dUTP for the 18S rDNA, according to the manufacturer’s manual. Telomeric (TTAGGG)n sequences were also mapped using the DAKO Telomere PNA FISH Kit/FITC (DAKO, Glostrup, Denmark). FISH experiments followed the methodology described in Yano et al. [31 ].

Two tandemly-arrayed DNA sequences isolated from the genome of an Erythrinidae species, Hoplias malabaricus, previously cloned into plasmid vectors and propagated in competent cells of Escherichia coli DH5α (Invitrogen, San Diego, CA, USA), were used. The first probe contained a 5S rDNA repeat copy and included 120 base pairs (bp) of the 5S rRNA transcribing gene and 200 bp of the nontranscribed spacer (NTS) [60 (link)]. The second probe corresponded to a 1400 bp segment of the 18S rRNA gene obtained via PCR from nuclear DNA [61 (link)]. These probes were directly labeled with the Nick-Translation mix kit (Roche, Manheim, Germany). The 5S rDNA was labeled with Spectrum Green-dUTP, and the 18S rDNA was labeled with Spectrum Orange-dUTP (Vysis, Downers Grove, IL, USA), according to the manufacturer’s manual. The small repetitive sequences (CA)15, (GA)15, (CAT)10, and (CGG)10 were directly labeled with Cy-3 (with the exception of (GA)15 which was direct labeled with FITC) during the synthesis, as described by [62 (link)]. Telomeric (TTAGGG)n sequences were also mapped using the DAKO Telomere PNA FISH Kit/FITC (DAKO, Glostrup, Denmark).