Golden view

The entire genomic sequence of the CDPK2 genes from individual T. gondii strains was amplified using the oligonucleotide primers FCDPK2 (forward primer: 5'-ATG CCG CTC AAG ACT TCC TGG CAT T-3') and RCDPK2 (reverse primer: 5'-TTA CCC CGT AGC GCG AGG CGT CAG ACT G-3'). The amplification reaction was carried out in a 25-μL total volume, including 12.5 μL Premix Ex Taq (TaKaRa Bio, Otsu, Shiga, Japan), 0.2 μM of each primer, and 100-200 ng template DNA. Amplification of DNA samples from individual strains was carried out in a thermocycler (Bio-Rad, Hercules, CA, USA) under the following conditions: denaturation at 94°C for 10 min (initial denaturation), followed by 35 cycles of 94°C for 50 s (denaturation), 67°C for 40 s (annealing), 72°C for 3 min and 40 s (extension), and a final extension step at 72°C for 10 min. The successful PCR amplifications were confirmed by electrophoresis on a 1% (w/v) agarose gel, and stained with GoldenView™ and the DL 5000 marker (TaKaRa) to estimate the sizes of the CDPK2 PCR products.