Thermal cycler

The Pf-PAN Rapid diagnostic kit (SD Biosensor, India) was used for the detection of P. falciparum and other Plasmodium infections using 20 μl of blood sample at the study site. Genomic DNA from all RDT positive samples was extracted from blood using QIAamp^® DNA Blood Mini Kit (QIAGEN, Germany) following the manufacturers’ manual. The DNA extracted was further subjected to species-specific nested PCRs as described by Snounou et al. [27 (link)]. The PCR was carried out in a Thermal cycler (Agilent, UK) and the PCR products, as well as RFLP sites/fragments, were subjected to 2.0% agarose gel electrophoresis followed by staining with 0.5% ethidium bromide. The species-specific bands (P. falciparum: 205 bp, P. vivax; 120 bp, Plasmodium malariae; 144 bp, Plasmodium ovale; 800 bp) were visualized and recorded digitally using the Gel-Doc system (Alpha Imager, USA).

Putative transformed shoots were screened for presence of Npt II gene by PCR. The 790 bp sequence of Npt II gene was amplified using gene specific primers listed in Table 1. Each PCR reaction was done in 20 µl reaction consisting of 2 µl of 10× reaction buffer, 100 ng genomic DNA, 10 mM dNTPs, 10 µM of each primer, and 1 U of Taq polymerase (Genet bio, Korea).The amplification reaction was performed in a thermal cycler (Agilent Technologies, USA) under the following conditions: initial denaturation 95 °C for 5 min, then 35 cycles of denaturation 95 °C for 30 s, annealing 56 °C and 60 °C for 30 s and elongation 72 °C for 1 min and final elongation 72 °C for 10 min. The amplified product was electrophoresed on 1% agarose gel and photographed through gel documentation system (Alpha imager).