Tae buffer tris acetate edta

DNA concentration and purity were estimated using DeNovix DS-11 FX (DeNovix Inc., Wilmington, United States). The concentration of extracted DNA was assessed by measuring the absorbance of the samples at A260 nm. Quality/purity was determined by analysing the A₂₆₀/A₂₈₀ ratio. DNA integrity was evaluated by electrophoresis in a 0.8% (w/v) agarose gel (NZYtech, Lisboa, Portugal) in 1 × TAE buffer (Tris-acetate-EDTA) (NZYtech, Lisboa, Portugal) stained with Green®Safe Premium nucleic acid stain (NZYtech, Lisboa, Portugal), and visualized under UV light using a Gel Doc XR+ (Bio-Rad Lab, Hercules, United States) and Quantity One software^®.

To validate the presence of amplifiable DNA in all samples, a species-specific PCR targeting the mitochondrial D-loop region producing a fragment of approximately 531 bp was performed using the respective set of forward and reverse primers sequences: FW 5′-AAC CCT ATG TAC GTC GTG CAT-3′ and RV 5′- ACC ATT GAC TGA ATA GCA CCT-3’ (Montiel-Sosa et al., 2000 (link)). The PCR reactions were performed in a final volume of 25 μL, containing 2 × NZYTaq II Green Master Mix (NZYtech, Lisboa, Portugal), 0.25 μM of each primer and 20 ng/μL of DNA. A negative and positive control was included in each assay. The reactions were incubated at 95°C for 5 min; followed by 35 cycles of 95°C/30 s, 50°C/30 s, 72°C/30 s, and a final 10 min of extension at 72°C. All runs included internal positive (swine sausage) and negative (fennel leaf) controls. All amplifications steps were performed on a Veriti™ Dx 96-well Thermal Cycler (Thermo Fisher Scientific, Massachusetts, United States). PCR products were separated by electrophoresis in 1.5% agarose gel in 1 × TAE buffer (Tris-acetate-EDTA) (NZYtech, Lisboa, Portugal) stained with Green®Safe Premium nucleic acid stain (NZYtech, Lisboa, Portugal).