Fast taq

At the IZSSA, genomic DNA was extracted from all 261 isolates and Type/Reference Strains (T/RS) as described previously [12 (link)]. Species identification was based on PCR amplification of the glyceraldehyde-3-phosphate dehydrogenase gene (gap) gene [20 (link), 21 (link)]. The primers GF-1 (5’-ATGGTTTTGGTAGAATTGGTCGTTTA-3’) and GR-2 (5’-GACATTTCGTTATCATACCAAGCTG-3’) were used for staphylococci whereas the primers Strept-gap-F (5’-ACTCAAGTGTACGAACAAGT-3’) and Strept-gap-R (5’-GTCTTGCATTCCGTCGTAT-3’) for streptococci. PCR was performed in a reaction mixture containing 2.5 µL 10 × reaction buffer, 1.5 µL dNTPs 1.25 mM, 1 µL of each primer (25 pmol each), 1 µL DNA template, 0.5 µL Fast Taq (Roche, Basel, Switzerland), and distilled water up to 25 µL. Reactions were carried out in an automated DNA thermal cycler (GeneAmp 9700, Applied Biosystems, CA, USA). Amplification conditions: initial denaturation for 5 min at 95 °C followed by 30 cycles of 1 min at 95 °C, 1 min at 50 °C (for staphylococci)/1 min at 54 °C (for streptococci), and 1 min at 72 °C with a final extension step of 10 min at 72 °C. The 933-bp (staphylococci) and 945-bp (streptococci) amplicons were examined by electrophoresis in 1% agarose gels, stained with Sybr^® Safe DNA gel stain (Invitrogen, CA, USA), and visualized under a UV transilluminator.

It has been previously described and shown in Table 1 about what the methylation and unmethylation-sensitive primers of LOC134466 and TAC1 were used in the study. In a 25 μl reaction mixture, we amplify 1.5 μl of bisulfite-converted DNA. The reaction mixture contained 200 μM dNTPs, 10X reaction buffer, 2.5 mM MgCL₂, 10 pM forward and reverse primers, as well as 1 U of FastTaq (Roche, Basel, Switzerland). Bisulfite-modified DNA was then amplified with two primer sets that differentiate methylated from unmethylated DNA. Meanwhile, we performed Hot-start PCR at an annealing temperature of 60 °C using hot-start Taq DNA polymerase (Thermo Fisher Scientific). Cases in which methylated alleles were present were repeated once again for confirmation.