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.
Dekaprime kit
Manufactured by Thermo Fisher Scientific
Sourced in Lithuania
The DekaPrime kit is a laboratory equipment product manufactured by Thermo Fisher Scientific. It is designed to perform a specific function in the laboratory environment. The core purpose of the DekaPrime kit is to provide an essential tool for researchers and scientists, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
4 protocols using dekaprime kit
1
Quantifying 5S rDNA Copy Number
2
Quantification of 5S rDNA Copy Number
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The procedure followed the protocol described by [38 (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 labelled with the 32P-dCTP (DekaPrime kit, Fermentas, Lithuania). The probe was hybridised at high stringency conditions (washing 2x SSC, 0.1% SDS followed by 0.1xSSC, 0.1% SDS at 65 °C). The hybridisation signals were visualised 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 genes was estimated using slot blot hybridisation. Briefly, the DNA concentration was estimated spectrophotometrically at OD260nm using 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 a serial dilutions of unlabelled plasmid inserts corresponding to the 5S monomers (GenBank KX965715-6), were 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 hybridisation conditions and visualisation of signals were the same as described above.
3
Southern Blotting of rDNA Probes
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Southern blotting followed the protocol described by Koukalova et al. (2010) (link) using rDNA probes, A-genome-specific IGS probe (IGS-A) and C-genome-specific IGS probe (IGS-C) labelled with 32P (DekaPrime kit, Fermentas, Lithuania). The hybridization signals were visualized by Phosphor imaging (Typhoon 9410, GE Healthcare, PA, USA) and signals were quantified using ImageQuant software (GE Healthcare).
4
5S rDNA Fluorescent In Situ Hybridization
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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 0.1xSSC, 0.1% SDS at 65 °C). The hybridization signals were
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 0.1xSSC, 0.1% SDS at 65 °C). The hybridization signals were
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