Nanodrop model 2000c

Participants drool saliva was collected in a DNA/RNA shield-saliva collection kit (Genesee Inc.). Genomic (g) DNA from the saliva was extracted using a spin column-based DNA isolation kit (Zymo Quick-DNA Miniprep Kit Cat# D4069). Extracted gDNA was quantified via spectrophotometry (NanoDrop Model 2000C, Thermo Fisher Sci.) and sample purity was estimated by absorbance ratio of A260/A280 (sample range: ≥1.8–2.0). Extracted DNA was diluted to 1ng/ul concentration and used as templates in real time quantitative polymerase chain reaction (PCRs; QuantStudio3; Applied Biosystems). A 5’ to 3’ exonuclease assay in TaqMan (Applied Biosystems) was used to amplify the gene SNP of interest. SNP genotyping calls were performed with TaqMan Genotyper Software (Thermo Fisher Sci. Inc). Human DNA samples with known genotype from Coriell Institute’s Medical Research Repository were used as control identifier for TaqMan Genotyper Software.

In order to evaluate the capacity of phage BrSP1 to control P. aeruginosa, we carried out “in vitro” experiments with strains Lfar01, ATCC 27853 and BOIJ02. TSB medium supplemented with CaCl2 (40 mg/L) and MgSO4 (40 mg/L) was used in these analyses. The phage concentration we used were either 10⁶ or 10⁷ plaque forming units per mL of bacterial culture media (PFU/mL). Test tubes with 10 mL of P. aeruginosa cells at early exponential phase were inoculated with phage BrSP1 and cultured at 36 °C in a benchtop incubator shaker (model Classic C24, New Brunswick Scientific®) at 120 rpm for 24 h. We removed samples from the culture at the time of viral inoculation (t = 0) and at 2, 4, 6, 8, 10, 12, and 24 h post-infection (h p.i.) (t = 2, t = 4, t = 6, t = 8, t = 10, t = 12, and t = 24, respectively). Control tubes were inoculated with SM buffer. We evaluated cell concentrations at each of these times measuring the absorbance at 600 nm (OD₆₀₀) (NanoDrop model 2000c, Thermo Scientific®) (Additional file 8). Each set of assays was done in triplicate.

In this experiment, the nSe-associated variations in the expressions of target genes were addressed in leaves (before RNA extraction, leaves were kept in −80 °C). RNA was purified from leaves that were well-grounded in liquid nitrogen using DEPC Water (Bio Basic, Canada), triazole (GeneAll Biotechnology Co, South Korea), and Dnase I (Fermentase, USA). Then, the accuracy of RNA extraction was verified using a Nanodrop (Thermo Scientific^™NanoDrop Model 2000c). Next, the synthesis of complementary DNA (cDNA) was carried out using a thermocycler (PEQLAB, 96Grad). In Table 2, the designed forward and reverse sequences of primers for Phenylalanine Ammonia Lyase (McPAL)- (XM_022284778), 4-Coumaroyl CoA ligase (Mc4CL)- XM_022281848, transcription Factor WRKY1 (NW_019104495), and Elongation factor (a housekeeping gene) are depicted. After that, the transcription rates of the target genes were estimated according to common SYBR green (GeneAll, South Korea) and the real-time quantitative PCR procedure (Applied Biosystems StepOne^™ Real-Time PCR). The delta CT protocol was utilized to calculate the expression rates presented as a fold difference.