Taq dna polymerase

Presences of various uropathogenic O-Serogroups (O1, O4, O2, O7, O6, O15, O8, O21, O25, O16, O22, O75, O18, and O83) in UPEC strains were investigated using the PCR techniques. Used primers for O- Serogroups amplification is shown in Table 1. The PCR methods for amplification of O1, O7, O6, O21, O16, and O75 Serogroups was performed with a total volume of 50 µL including 2.5 mM MgCl2, 0.4 µM of forward primer, 0.4 µM of reverse primer, 0.4 µL PCR buffer 10X, 300 µM dNTP (Fermentas), 2 U Taq DNA polymerase (Fermentas), and 3 µL DNA template. The DNA was then amplified by 30 successive cycles of denaturation at 95°C for 30s, primer annealing at 55°C for 60s, and DNA chain extension at 72°C for 60s. Also, The PCR methods for amplification of O2, O4, O15, O18, O22, O25, and O83 serogroups was performed with a total volume of 50 µL including 2.5 mM MgCl2, 0.6 µM of forward primer, 0.6 µM of reverse primer, 0.4 µL PCR buffer 10X, 300 µM dNTP (Fermentas), 1.5 U Taq DNA polymerase (Fermentas), 3 µL DNA template, and 3 µL DMSO. The DNA was then amplified by 30 successive cycles of denaturation at 94°C for 60s, primer annealing at 56°C for 60s, and DNA chain extension at 72°C for 90s. The programmable thermal cycler (Eppendorf, Mastercycler® 5330, Eppendorf-Netheler-Hinz GmbH, Hamburg, Germany) PCR device was used in all the PCR reactions.

Viral RNA was obtained from serum samples using a commercial kit (QIAamp Viral RNA Mini Kit, QIAGEN). Then, the cDNA was synthesized using the primer 1302D [22 (link)] 0.67 µM and the enzyme RevertAid RT (Thermo Scientific) following the suggested protocol. Amplification conditions and cycling were modified from protocols previously published [22 (link)]. The first round was carried out with 1× buffer, 2 mM MgCl₂, 0.2 mM dNTPs (Promega), 0.5 µM using the primers 8531U, and 1302D [22 (link)] (Supplementary Table S1) and Taq DNA polymerase (Thermo Scientific) 1.25. There were cycles of 95 °C for 2 min, followed by 35 cycles of 95 °C for 30 s, 56 °C for 30 s, extension at 72 °C for 1 min, and 72 °C for 10 min. The second round reaction was carried out with 1× buffer, 2 mM MgCl₂, 0.2 mM dNTPs (Promega), using the primers HDV-E and HDV-A at 0.5 µM [22 (link)], and Taq DNA polymerase (Thermo Scientific) 1.25. There were cycles of 95 °C for 2 min, followed by 35 cycles of 95 °C for 30 s, 56 °C for 30 s, extension at 72 °C for 1 min and 72 °C for 10 min.

All isolates testing positive for AmpC β-lactamase production on the CC-DDST were tested by polymerase chain reaction (PCR) for pAmpC gene carriage. Further, as pAmpC β-lactamase genes have also been detected in isolates with reduced susceptibility to third-generation cephalosporins, we tested isolates with inhibition zone diameters of ≤27 mm, ≤25 mm and ≤22 mm for cefotaxime, ceftriaxone and ceftazidime, respectively, for pAmpC gene carriage
^{17 ,
23 (link)}. In-house multiplex PCRs targeting AmpC β-lactamase genes
bla_CIT, bla_DHA, bla_MOX, bla_FOX, bla_EBC,
bla_ACCand
bla_CMY-2 were performed using published primers, Thermo-Fisher Taq DNA polymerase and PCR master-mixes, and conditions
^{6 (link),
26 (link)}. Amplification was performed in a 3Prime Mid-size thermocycler (Techne, UK) and the expected amplicon sizes were successfully generated. PCR amplification of ESBL genes
bla_CTX-M,
bla_TEM and
bla_SHV was performed with Taq DNA polymerase (Thermo-Fisher Inc.) using published primers
^{27 (link)}. Amplicons were sequenced (ACGT, Wheeling, IL, USA) by the chain termination method (Sanger sequencing) and sequences confirmed through
BLAST-searching at NCBI. Phylogenetic group typing of
E. coli was done according to the method of Clermont et al, in which PCR of a combination of two genes (
chuA &
yjaA) and an anonymous DNA fragment are used to classify strains
^{28 (link)}.

T. brucei brucei Lister 427 PCF collected by centrifugation were washed twice with PBS (0.1 M sodium phosphate, 0.15 M NaCl, pH 7.4) and genomic DNA was isolated using DNAzol Reagent as described by the manufacturer (Life Technologies, Carlsbad, CA, USA).
Polymerase Chain Reaction (PCR) was performed at a final volume of 50 μl containing genomic (~300 ng) or plasmid DNA (~50 ng), 30 pmoles of the specific primers (Macrogen, Seoul, Korea), 2.5 mM of each deoxynucleotide triphosphate (dATP, dCTP, dGTP, dTTP) (New England Biolabs), 1.5 mM MgCl₂ (Life Technologies), 0.2 UI Taq DNA polymerase (Life Technologies) and reaction buffer as indicated by the manufacturer (Life Technologies).
For the amplification reaction a thermocycler (Mycycler, Bio-Rad, Hercules, CA, USA) was employed: an initial denaturation step at 94°C for 10 min was followed by 35 cycles of a) denaturation at 94°C 1 min, b) hybridization at 55°C 1 min, c) elongation at 72°C -1 min per kb of DNA amplified; the final elongation step was performed at 72°C for 10 min.