Taq buffer with nh4 2so4

After third day of inoculation, subcultures were centrifuged at 12000 gr for one minute and genomic DNA was isolated with DNAzol kit (Thermo Fisher Scientific, Waltham, MA USA) from pellets according to the manufacturer’s instructions. The Blastocystis barcode region of SSU-rDNA gene was amplified in a single PCR with the primers RD5 and BhRDr (15 (link)) and with a gel documentation system (Infinity, Vilber Lourmat). The reaction was set in a 30-μl volume containing: 1–2 μl of template DNA, 0.4 pmol of each of the primers, 0.3 U of Taq DNA polymerase (Fermentas), 0.2 mM of each dNTP (Fermentas), 1× Taq buffer with (NH₄)₂SO₄ (Fermentas). PCR amplifications were purified and sequenced by a commercial facility (MedSanTek, Istanbul) by using Applied Biosystems 377 DNA Sequencer.

Two positive (E. coli SE80003 and E. coli 3682) and one negative control (E. coli K5-23) were used in fumC SNPs detection PCR. Amplification of fumC was performed by singleplex PCR using one set of primers. PCR was run with a 25 µl reaction mixture containing 2 µl DNA template, 1 µl of a set of primers), 2.7 µl of 10X Taq Buffer with (NH₄)₂SO₄, 3.7 µl MgCl₂, 0.5 µl of dNTPs and 0.3 µl Taq polymerase (Fermentas, Germany).
The PCR conditions used were as follows: 95 °C for 5 min; 30 cycles of denaturation at 94 °C for 1 min; annealing at 55 °C for 1 min and initial extension at 68 °C for 3 min. A final extension of 72 °C was run for 10 min.