Dntps

Simplex PCRs, carried out for Pfcrt, were performed using a mastermix containing 5 µl of Q5 Reaction Buffer (New England BioLabs), 0.5 µl of dNTPs (1n nM stocks, New England BioLabs), 0.25 µl Q5 Hot Start High-Fidelity DNA Polymerase (New England BioLabs), and 15.75 µl Milli-Q water (Merck). For each reaction, a total of 1.25 µl of forward and 1.25 µl of reverse primer (10 pmol/µl stocks) were used with 1 µl of DNA for a total reaction volume of 25 µl. Multiplex PCRs (combinations described in Supplementary Table 2) were performed using a mastermix containing 5 µl of Q5 Reaction Buffer (New England BioLabs), 0.5 µl of dNTPs (1n nM stocks, New England BioLabs), 0.25 µl Q5 Hot Start High-Fidelity DNA Polymerase (New England BioLabs), and 15.8 µl Milli-Q water (Merck). For each multiplex reaction, 0.6 µl of both forward primers, for a total of 1.2 µl of forward primer, and 0.6 µl of each reverse primer, for a total of 1.2 µl of reverse primer, were used with 1 µl of DNA for a total reaction volume of 25 µl. The reactions were carried out in a thermocycler consisting of the following steps: Heat activation for 15 min at 72 °C, 30 cycles of denaturation for 20 s at 95 °C, annealing for 2 min at 55 °C, elongation for 2 min at 72 °C, and a final elongation for 10 min at 72 °C, followed by a hold at 10 °C.

cDNA synthesis was performed as described in Balcells et al. [32 (link)], with the following reagents: 10ng of total RNA, Poly-A polymerase (NEB: M0276L), MulV RT enzyme (NEB M0253L) and dNTPs (NEB N0447L). The cDNA was diluted 10-fold for the RT-qPCR. The qPCR reaction was performed in RealQ Plus 2x Master Mix Green (without Rox, Ampliqon, A323406), with cycling conditions: 1x 95°C for 15 minutes, 40 cycles of (95°C for 30sec and 60°C for 30sec) using a Light Cycler 480 system (Roche).