5 primestar gxl buffer

The fragment of the target gene was amplified with PCR from the cDNA of T. annulata-infected cells. The specific primers were designed based on the reference sequence of SVSP950454 (Accession No: XM950454, SVSP454), and the nucleotide sequences were: SVSP454-F (5′-CCGGAATTCATGAATAAATATATAACATAT-3′) and SVSP454-R (5′-TGCACTGCAGATCCTCCGATTTATCATATTT-3′) with the restriction site underlined. The final volume of PCR reaction was 50 μL, which contained 10 μL 5× PrimeSTAR GXL Buffer (TaKaRa, Dalian, China), 4 μL dNTP Mixture (2.5 mM), 20 μM of each primer, 1 μL PrimeSTAR GXL DNA Polymerase (TaKaRa, Dalian, China), 4 μL of cDNA template, and 17 μL ddH₂O. The protocol of PCR reactions was performed as follows: 35 cycles of denaturation (98 °C for 10 s, 50 °C for 15 s, 68 °C for 20 s) and the final extension at 68 °C for 10 min. The PCR products were purified by using a Cycle-Pure Kit (OMEGA Bio-Tek, USA), which was followed by digestion with restriction enzymes EcoRI and PstI (Thermo Fisher Scientific, #FD0274 and #FD0614). The products were then ligated to the pGBKT7 plasmid, which was digested with the same enzymes as previously described. Finally, the recombinant SVSP454-pGBKT7 plasmid was further identified using double restriction enzyme digestion and sequencing (Sangon Biotech, Shanghai, China), and only the positive plasmid was used as the bait plasmid for further experiments.