Nick translation mix kit

L. multiflorum, F. pratensis and F. glaucescens genomic DNA was sonicated for 5 min in an ultrasonic bath, checked on 1% agarose gel, and the DNA samples, sheared to 500–2500 bp fraction, were chosen for labelling. DNA was labelled with rhodamine-11-dUTP (Roche) or fluorescein-12-dUTP (Roche) by nick translation, using Nick Translation Mix kit (Roche). L. multiflorum rhodamine labelled probe was used specifically to highlight simultaneously both L. multiflorum (red) and F. pratensis (mauve), as described by Pašakinskienė et al. [30 (link)]. The pTA71 plasmid containing wheat 45S ribosomal DNA repeats was cleaved with the EcoRI restriction enzyme to release the insert and labelled with fluorescein-12-dUTP by nick translation.

Two tandemly arrayed DNA sequences isolated from the genome of Hoplias malabaricus, previously cloned into plasmid vectors and propagated in competent cells of Escherichia coli DH5α were used as probes (Invitrogen, San Diego, CA, USA). The first probe corresponded to the 5S rRNA coding region, comprising 120 base pairs (bp) associated with a non-transcribed spacer, NTS [47 ], labeled with the Nick-Translation mix kit (Roche, Manheim, Germany) using the SpectrumOrange-dUTP (Vysis, Downers Grove, IL, USA). The second probe corresponded to a 1,400 bp segment of the 18S rRNA gene [48 (link)], also labeled by means of Nick-Translation but using the SpectrumGreen-dUTP (Vysis, Downer Grove, IL, USA). FISH was performed under high stringency conditions following the protocol described in [18 (link)].

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).