Veriti dx thermal cycler

Germplasm evaluation on agronomically important traits, was addressed by investigating for the presence of alleles, that control low alkaloid content, resistance to anthracnose and vernalization requirement, applying a set of recently developed, specifically designed dCAPS molecular markers (Table 4) [18 (link),19 (link),48 (link)]. PCR amplification reactions were carried out on Veriti Dx Thermal Cycler (Applied Biosystems^®, Waltham, MA, USA), using KAPA Taq ReadyMix PCR Kit (Kapa Biosystems, Wilmington, MA, USA) in a total reaction volume of 15 μL, containing 20 ng genomic DNA, 1X KAPA Taq ReadyMix Mix, 0.2 μM of each primer, 1.5 mM MgCl₂, and PCR-grade H₂O. PCR amplification was performed under the following conditions: initial denaturation at 94 °C for 4 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing for 30 s, extension at 72 °C for 30 s, and final extension at 72 °C for 4 min. In order to detect the presence of the target allele of each marker, all samples were subjected to post-PCR restriction enzyme digestion, following the appropriate protocol provided by New England BioLabs (Ipswich, MA, USA). The presence or absence of the target alleles was then verified by 3% agarose gel electrophoresis (1× TAE) on 100 volts for 40 min.

Primers designed by Conrad et al. [10 (link)] were used for detection of stx₁ and stx₂ (Table 1). The reactions were performed as follows: 95°C for 5 min, followed by 35 cycles of 94°C for 30 s, 60°C for 45 s, 72°C for 90 s, and a final extension of 72°C for 5 min. Conrad et al. [10 (link)] primers were also used for detection of serogroups (O26, O45, O103, O121, O145 O157; Table 1). PCRs contained a final volume of 25μL and 0.2 μM each primer, 1x HotStar Taq Plus MasterMix (Qiagen® Hilden, Germany), 1x Coral Load PCR buffer, 2 μL DNA template, and nuclease-free water. The reactions were performed in a Veriti™ Dx Thermal Cycler (Applied Biosystems). To ensure that the PCR primers used were not responsible for inconsistent stx₁ and stx₂ results, virtual PCR was performed for the 50 isolates using Geneious 10.2.6 software (Biomatters, Auckland, Australia) to compare primers of Scheutz et al. [22 (link)] and Conrad et al. ([10 (link)]; Table 1). Also, two base pair (bp) mismatches between primer and sequences for both stx and serogroup were allowed to ensure that inconsistences which can lead to amplification were considered. For other configurations default parameters were used.

A total of 500 ng RNA was reverse transcribed using ThermoFisher Superscript IV Vilo Kit (catalog #11756500, ThermoFisher Scientific, Waltham MA) following manufacturer’s instruction on an Applied Biosystems VeritiDx Thermal Cycler. Each RT reaction was based on a total of 500ng RNA in a volume of 20μL. Multiple reactions for the same sample were combined in one tube.

Droplet digital PCR was carried as previously described [22 (link), 23 (link)] and briefly detailed hereafter. The ddPCR mixture contained 7,5 μl of 2× ddPCR Evagreen Supermix (Bio-Rad, Hercules, USA), 10 nM of the testing forward and reverse primers or GAPDH primers and 5 ng of cDNA (RNA equivalent) in each 15 μl reaction. The 15 ul reaction was placed in the droplet generator (Bio-Rad #186–3002) resulting in around 20,000 individual droplets [22 (link)]. Sealed plates with the droplets were cycled (Veriti DX thermal cycler, ThermoFisher Scientific) under the following conditions: 2 min at 30 °C; 10 min hold at 95 °C; 48 cycles of 95 °C for 50s then 59 °C for 120 s; 5 min at 4 °C, 5 min at 90 °C. After amplification, the plate with the droplets was read on a Bio-Rad droplet reader (QX200) and data was analysed with the Quantasoft software.

Droplet digital PCR (ddPCR) was carried out according to manufacturer recommendations and as described^{43 (link), 44 (link)}. Briefly, the ddPCR mixture contained 7.5 μl of 2× ddPCR Evagreen Supermix (Bio-Rad, Hercules, USA), 10 nM of both the Zeb2-NAT, Zeb2 intron 1, or GAPDH forward and reverse primers, and 5 ng of cDNA (RNA equivalent) in each 15-μl reaction. The 15-μl reaction was assembled into a droplet cartridge (Bio-Rad, Hercules, USA), according to the Bio-Rad protocol and the cartridge was placed in the droplet generator (Bio-Rad #186-3002) giving rise to around 20,000 individual droplets in an emulsion^{43 (link)}. Droplets were transferred to a 96-well plate (Eppendorf, Hamburg, Germany) and thermal sealed with a foil lid (Eppendorf, Hamburg, Germany). Sealed plates were cycled using a Veriti DX thermal cycler (ThermoFisher Scientific) under the following conditions: 2 min at 30 °C; 10-min hold at 95 °C; 48 cycles of 95 °C for 50 s and then 59 °C for 120 s; 5 min at 4 °C; and 5 min at 90 °C. After amplification, the plate was transferred to a Bio-Rad droplet reader (QX200) from which data were extracted and analyzed with the Quantasoft software following manufacturer recommendations.