Vacuum slot blotter

The procedure followed the protocol described by Koukalova et al. (2010) (link). The 5S rDNA probe was a 243 bp insert of the 5S_Eci_a clone (GenBank KX965716) from E. cisalpinus. The plasmid insert was amplified and labeled with the 32P-dCTP (DekaPrime kit, Fermentas, Lithuania). The probe was hybridized at high stringency conditions (washing 2x SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS at 65°C). The hybridization signals were visualized by Phosphor imaging (Typhoon 9410, GE Healthcare, PA, USA), and signals were quantified using ImageQuant software (GE Healthcare, PA, USA). The copy number of 5S rDNA genes was estimated using slot blot hybridization. Briefly, the DNA concentration was estimated spectrophotometrically at OD260nm using a Nanodrop 3300 fluorospectrometer (Thermo Fisher Scientific, USA). Concentrations were verified by the electrophoresis in agarose gels using dilutions of lambda DNA as standards. The three dilutions of genomic DNA (100, 50, and 25 ng), together with serial dilutions of unlabeled plasmid inserts corresponding to the 5S monomers (GenBank KX965715-6). Each aliquot was denatured in 0.4 M NaOH and blotted onto a positively charged Nylon membrane (Hybond XC, GE Healthcare, USA) using a vacuum slot blotter (Schleicher-Schuell, Germany). The probe and the hybridization conditions and visualization of signals were the same as described above.