Mastercycler ep gradient thermocycler

For PCR, genomic DNA was extracted from conidia with the DNeasy 96 Plant Kit (Qiagen) as already described (Daverdin et al., 2012 (link)). Standard PCRs were performed in an Mastercycler EP gradient thermocycler (Eppendorf) as described in Daverdin et al. (2012 (link)). Sequencing was performed using a CEQ 8000 automated sequencer (Beckman Coulter) according to the manufacturer's instructions.
Total RNA was extracted from infected cotyledons of B. napus or B. oleracea genotypes using TRIzol reagent (Invitrogen) according to manufacturer's protocol. Total RNA was treated with RNase‐free DNase I (New England Biolabs).

DNA from EBV was identified using the set of primers EBNA1-F 5′-AGG CCA TTT TTC CAC CCT G-3′ and EBNA1-R 5′- CTT CTC CTG GGT CAT CTG CG-3′, which amplify a fragment of 114 bp from the EBNA1 gene. Detection of HCMV DNA was performed using the set of primers forward UL44-F 5′-GTG CGC TCA AGG AGA ACA C-3′ and reverse UL44-R 5′-TGC ACG TAG AAC TTG GTC AG-3′, that amplify a 181 bp region from the UL44 gene. PCR reactions were performed in a total volume of 20 μL containing 200 ng DNA, 4 mM MgCl₂, 0.2 mM dNTP's, 5 pmol of each primer for EBNA1, 0.4 μM of each primer for UL44 and 1 U Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA). All PCR reactions were run in a Mastercycler EP gradient thermocycler (Eppendorf, Hamburg, Germany). Cycling conditions included 1 cycle of initial denaturation at 94°C for 5 min, followed by 40 cycles of 94°C for 20 s, 57°C for 20 s for EBNA1 gene (58°C for UL44), 72°C for 30 s and a final extension cycle at 72°C for 7 min. All PCR products were resolved in 2.5% agarose gels, stained with ethidium bromide and visualized under UV light. Samples were considered positive for EBV or HCMV when the PCR products of the corresponding sizes were identified. As a positive control, an EBV⁺/HCMV⁺ sample was used. The specificity of viral DNA in the sample was verified by sequencing.