Midori green advance dye

Genomic DNA was isolated from 6-week-old callus using the CTAB method according to the protocol by Rogers and Bendich⁸⁶ with modifications as described by Klimek-Chodacka et al.^{87 (link)}. The PCR reaction was conducted in a Mastercycler gradient thermocycler (Eppendorf, Hamburg, Germany) under the following conditions: the initial denaturation for 3 min at 94 °C, 30 cycles of 30 s at 94 °C, 30 s at 56 °C and 30 s at 72 °C, followed by the final extension for 3 min at 72 °C. The 10 μL of reaction mixture included 50 ng/μL DNA, 0.1 μM of each primer (Table 1) and 2 × buffer including Taq polymerase and dNTPs (PCR Mix Plus, A&A Biotechnology, Gdynia, Poland). The amplified products were visualised using the MidoriGreen Advance dye (Nippon Genetics) after electrophoresis in 1.5% agarose gel.

The restriction enzymes NdeI, BamHI, and T4 DNA ligase were purchased from Thermo Scientific (Mannheim, Germany), and the Promega Corporation (Madison, WI, USA). The GeneJET Gel Extraction Kit was obtained from Thermo Scientific (Hudson, NH, USA). Profinity IMAC Resins and Gel Filtration Standard, used as a lyophilized mixture of molecular-weight markers, ranging from 1350 to 670,000 Da, were purchased from Bio-Rad (Hercules, CA, USA). Isopropyl β-d-1-thiogalactopyranoside (IPTG), Tris-HCl, PMSF, β-mercaptoethanol, glycerol, NaCl, MgCl₂, imidazole, dithiothreitol (DTT), Coomassie brilliant blue R, G6P, NADP⁺ substrates, ampicillin, and kanamycin antibiotics were obtained from Sigma-Aldrich (St. Louis, MO, USA). The Midori Green Advance dye was purchased from NIPPON Genetics Europe, Dueren, Germany.

The characterization of SE events in the genes chosen for the validation of DASs was conducted with the use of Labcycler 48s (Syngen Biotech, Poland) and StartWarm HS-PCR Mix (A&A Biotechnology, Poland). The reaction mixture, in a final volume of 25 µL, contained 12.5 µL of Hot Start PCR Mix, primers (forward and reverse), nuclease-free deionized water, and 30 ng of cDNA. In non-template control, cDNA was substituted by water, or the reverse transcription step was omitted. PCR conditions and the primer sequences of chosen DASs are detailed in Table 1. The obtained PCR products were analysed on 1.5% agarose gels containing Midori Green Advance dye (Nippon Genetics Europe, Germany).