Platinum superfi 2 high fidelity dna polymerase

According to the instruction manual, viral RNA was extracted using the E.Z.N.A Viral RNA Kit (OMEGA Bio-Tek). The cDNA was synthesized using Reverse Transcriptase M-MLV (Takara, Dalian, China) and Random Primer (Takara, Dalian, China). We designed 12 and 10 pairs of specific primers for full-length genomic amplification of TGEV and PDCoV positive samples, respectively (Tables S1 and S2), by Platinum SuperFi II high-fidelity DNA polymerase (Invitrogen, Waltham, MA, USA). PCR amplification products were ligated using DNA A-Tailing Kit (Takara, Dalian, China) and pMD18-T vectors (Takara, Dalian, China), and finally, all plasmids were sent to Tsingke Biotechnology Co., Ltd. (Beijing, China). for Sanger sequencing.

An overnight culture of K. pneumoniae KP35 was prepared, and 1 ml was centrifuged at 8000 g for 6 min at 4 °C. Genomic DNA (gDNA) was extracted, purified and stored as described in the DNA and RNA extraction section (see below). Primers Kpn-2_Fw+Kpn-4_Rev (519 bp) and Kpn-4_Fw+Kpn-2_Rev (667 bp), and the Platinum SuperFi II high-fidelity DNA polymerase (Invitrogen), were used to amplify the attB and attP regions by PCR. Two rounds of PCR were carried out. For the first round, 50 ng gDNA was used as a template. For the second round, 1 µl of the reaction mix from the first round was used as a source of template. Each PCR round comprised 35 amplification cycles and used the same primers and polymerase. The amplification products were visualized by electrophoresis at 90 V for 55 min in TAE buffer, using 1 % agarose with 1× SafeView Plus (Fermelo Biotec). The PCR products were purified using the NucleoSpin gel and PCR clean-up kit (Macherey-Nagel), according to the manufacturer's instructions. The purified PCR products were sequenced (Sanger) in the Unidad de Secuenciación y Tecnologías Ómicas at Pontificia Universidad Católica de Chile (Santiago, Chile).