Uno 2 thermal cycler

In order to confirm the integration of the transgene into the hairy roots genome, total genomic DNA from 7 lines (SOPSS1-7) showing the best growth rate were isolated using Genomic Mini AX Plant kit (A&A Biotechnology) following the manufacturer’s procedures. Additionally, plasmid DNA (isolated using Plasmid Miniprep DNA purification kit, EURx, Gdansk, Poland) was also isolated from A. rhizogenes cells as a positive control. PgSS1 sequence specific primers (For-5′-gcatggaagagctttgacaac-3′, Rev-5′-atgggaagtttgggggcaat-3′). Amplification conditions: three minutes at 95 °C; 35 cycles of 30 s at 95 °C, 30 s at 52 °C, one minute at 72 °C; finally, five minutes extension at 72 °C. Final volume of all samples was 25 μL (1 μL of each primer (10 μM), 0.5 μL of DNA (50 ng/μL), 12.5 μL of PCR Mix Plus master mix (A&A Biotechnology, Gdynia, Poland) and 11 μL of PCR Ultra-Pure Water H2O (Blirt). DNA amplification was performed in a Biometra UNO II thermal cycler. The PCR products were then visualized on 1.5% agarose gel stained with ethidium bromide.

Specific primers were used to amplify sequences of 6 different virulence genes. Primer sequences and predicted sizes of the PCR products are shown in (Table 1). The amplification reactions were carried out using Biometra, UNO II thermal cycler (Goettingen, Germany) under the following conditions: initial denaturation at 95°C for 5 min, followed by 35 cycles of: 30 s at 94°C for, 30s at 63°C, then 30 s at 72°C, with a final extension step at 72°C for 5 min. PCR was performed in a 25 ml reaction mixture containing1 ul of template DNA (*100 ng/ml), 12.5 ml of PCR mastermix (Maxima Hot Start Green PCR Master Mix, USA), and 1 ul (10 pmol) of each primer and 9.5 ml of nuclease free water. PCR products were resolved on 2% agarose gel and visualized under a UV transilluminator (Biometra).