Maxima h minus first strand synthesis kit

Glutathione S-transferase (GST) fusion constructs were prepared to be able to produce PRK proteins in bacteria. For this purpose, a cDNA library was prepared from wild-type C. elegans samples using the Maxima H minus first strand synthesis kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. The prk-1 cDNA (isoform A, amino acids 1–530) was amplified from there with the prk-1 primers described in the previous section, and ligated into the pGEX-6P-1 plasmid (GE Healthcare) between BamHI and XhoI sites. Full-length prk-2 cDNA was subcloned from the rab-3:prk-2 plasmid (Zheng et al., 2011 (link); kindly provided by Michael Nonet, Washington University, St. Louis, MO), and ligated between EcoRI and SmaI sites in the pGEX-6P-2 plasmid. Preparation of GST-tagged human PIM1 (full-length short isoform) has been previously described (Santio et al., 2016 (link)), and GST-tagged human NFATC1 (amino acids 1–418) was a kind gift from S. N. Ho (Stanford University, Stanford, CA).

Expression of selected export genes were analyzed by RT-PCR with RNA isolated from the ring, trophozoite, and schizont stages. The total RNA was isolated using the TRIzol method (50 (link)) followed by purification with RNeasy kit (# 74104, Qiagen). The eluted RNA was used for cDNA synthesis. The cDNA synthesis was carried out using the Maxima H minus First-strand synthesis kit (Cat # K1681, Thermo Scientific). Afterward, cDNA synthesis for all the reactions was carried out by PCR using following conditions: 25 °C for 10 min followed by 50 °C for 15 min; reactions were terminated at 85 °C for 5 min. The qRT-PCR was carried out with freshly synthesized cDNA using SYBR Green mix (Thermo Scientific) (51 ).