Taq dna ligase reaction buffer

The DNA oligonucleotides were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) and purified by high-performance liquid chromatography. Taq DNA ligase (40 U μL⁻¹), 10 × Taq DNA ligase reaction buffer (200 mM Tris-HCl, 250 mM KAc, 100 mM Mg(Ac)₂, 100 mM DTT, 10 mM NAD, 1% Triton X-100, pH 7.6), Bst 2.0 DNA polymerase (8 U μL⁻¹) and 10 × ThermoPol reaction buffer (200 mM Tris-HCl, 100 mM (NH₄)₂SO₄, 500 mM KCl, 20 mM MgSO₄, 1% Tween-20, pH 8.8) were all purchased from New England Biolabs (Beijing, China). SYBR Green I was obtained from Generay Biotech. Co., Ltd. (Shanghai, China). DNA marker, 6 × loading buffer and dNTPs were purchased from Takara (Dalian, China). TIANamp Genomic DNA Kit was purchased from Tiangen Biotech Co., Ltd. (Beijing, China). The nuclease-free water was purchased from Thermo Fisher Scientific Inc (Vilnius, Lithuania) and used in all ligation reaction and ligation-initiated LAMP assays. All the reagents were of analytical grade and used without further purification. All solutions were prepared with ultrapure water from Millipore Milli-Q water purification system (Millipore, USA). The detailed oligonucleotide sequences were listed in Table S1 (Supporting Information).

The multiplex ligation reaction mix combined 1 to 5 nM of each of the 40 probes with 1X Taq DNA ligase reaction buffer (New England BioLabs), 2 to 6 units of Taq DNA ligase (New England BioLabs), and 2 or 4 μL of template DNA and was brought to a final volume of 10 μL with nuclease free distilled water (Life Technologies). The thermal cycling programme (Swift MaxPro, Esco) included 5 or 10 min of denaturation at 95°C followed by 30 cycles of 25 s at 94°C and 30 s at 58°C (Table 1: Multiplex oligonucleotide ligation). Each experiment included a positive control for the reaction and a no-template-control (NTC) to measure background signal for which the template DNA was replaced by, respectively, positive control template DNA and nuclease free distilled water. The results of neither the positive nor the negative control were taken into account for the statistical analysis and are not included in the figures shown, since these results are not representative of a bacterial isolate to be characterised.