A 681bp fragment of the D. gallinae mitochondrial cytochrome c oxidase subunit I (COI) gene was amplified using the primers COI1Fyuw114 (5′-AGATCTTTAATTGAAGGGGG-3′) and COI1Ryuw114 (5′- AAGATCAAAGAATCGGTGG-3′) corresponding to nucleotide positions 61 to 742 [GenBank accesion number AM921853 ; (19 (link))].
PCR was perfomed in a volume of 25 μl containing 12.5 μl 2x MyTaqTM (Bioline, London, UK), 400 pM of each primer (Sigma-Aldrich, Darmstadt, Germany) and 2 μl of DNA template. PCR cycling conditions were initial denaturing at 95°C for 5 min, followed by 35 cycles of denaturing at 95°C for 30 s, annealing at 55°C for 30 s, and elongation at 72°C for 30 s. Final elongation was performed at 72°C for 5 min. A T Gradient thermocycler was used (Biometra, Jena, Germany). After amplifcation, PCR products were resolved by electrophoresis in 1.0% (w/v) agarose gels, using 5X DNA loading buffer (Bioline, London, UK), Safeview Nucleic acid stain (NBS Biologicals, Cambridgeshire, UK). The GeneRuler 1 kb ladder (Thermo Fisher Scientific, Waltham, Massachusetts) was used to assess product size. Each PCR amplicon was purified using a Qiagen QIAquick PCR purifiction kit as recommended by the manufacturer (Qiagen GmbH, Hilden, Germany) and eluted in 30 μL dH2O.
PCR was perfomed in a volume of 25 μl containing 12.5 μl 2x MyTaqTM (Bioline, London, UK), 400 pM of each primer (Sigma-Aldrich, Darmstadt, Germany) and 2 μl of DNA template. PCR cycling conditions were initial denaturing at 95°C for 5 min, followed by 35 cycles of denaturing at 95°C for 30 s, annealing at 55°C for 30 s, and elongation at 72°C for 30 s. Final elongation was performed at 72°C for 5 min. A T Gradient thermocycler was used (Biometra, Jena, Germany). After amplifcation, PCR products were resolved by electrophoresis in 1.0% (w/v) agarose gels, using 5X DNA loading buffer (Bioline, London, UK), Safeview Nucleic acid stain (NBS Biologicals, Cambridgeshire, UK). The GeneRuler 1 kb ladder (Thermo Fisher Scientific, Waltham, Massachusetts) was used to assess product size. Each PCR amplicon was purified using a Qiagen QIAquick PCR purifiction kit as recommended by the manufacturer (Qiagen GmbH, Hilden, Germany) and eluted in 30 μL dH2O.
Free full text: Click here
