Kapa taq readymix kit

TMEV VP1 (1-112) was expressed in the E. coli strain JM109 from plasmid pCRTVP1. To create this plasmid, nucleotides 1-336 of the TMEV VP1 coding sequence were PCRamplified from the TMEV GDVII cDNA, using the KAPA Taq ReadyMix kit (KAPA Biosystems, Cape Town, South Africa) and gene-specific oligonucleotide primers. The PCR product was ligated into plasmid pQE-80L (Qiagen, Mannheim, Germany) by restriction with BamHI and SalI. To confirm the presence of the insert and correct open reading frame, plasmid pCRTVP1 was sequenced by Inqaba Biotechnical Industries (Pty) Ltd., Pretoria South Africa.

Plasmids containing the TMEV VP1 full length protein and VP1 truncations were constructed.
The sequence of TMEV VP1 was obtained by PCR of pGDVIIFL2, a plasmid that carries the full-length cDNA of TMEV GDVII (Fu et al., 1990) , using the KAPA Taq ReadyMix kit (KAPA Biosystems, South Africa). The forward primer NUVP1F and reverse primers NUVP1R, NU1-112R, NU1-195R and NU1-221R were used to generate the plasmids pVP1 (Full length), pVP1∆113-276, pVP1∆196-276, pVP1∆222-276 (C-terminal truncations) respectively. Primers NU159-276F and NU159-276R were used to generate pVP1∆1-158 (N-terminal truncation) (Table 1).
PCR cycles included an initial denaturation step at 95º C for 1 min, 30 cycles of: denaturation at 95º C for 1 min, annealing at 60º C for 1 min and elongation at 72º C for 1 min, followed by a final elongation step at 72º C for 7 min. The PCR products were ligated into the plasmid vector pQE-80L (Qiagen, Mannheim, Germany) by restriction with BamHI and SalI (ThermoFischer Scientific, USA). To confirm the presence of the inserts and correct open reading frames, all plasmids were sequenced by Inqaba Biotechnical Industries (Pty) Ltd., Pretoria South Africa.