Fusion high fidelity dna polymerase

The coding sequences of β-lactamase and DeepBL candidate genes were amplified from FK688 gDNA using Fusion High-Fidelity DNA polymerase (New England BioLabs) with the oligonucleotide primers listed in Table 6. The PCR products and the anhydrotetracycline (ATc)-inducible expression vector pJP-CmR were digested with restriction enzymes using either NcoI and HindIII, or EcoRI and HindIII (New England BioLabs) and ligated to create the plasmids listed in Table 5. Plasmids were verified by sequencing, transformed into E. coli BW25113 or K. quasipneumoniae FK688, and selected with chloramphenicol. Target gene expression was induced with 35 ng/mL ATc. The parental plasmid pJP-CmR was used as the control in all experiments.

16S PCRs were performed using the universal 16S rRNA bacterial primers 27F and 1392R (Table S2). Primers were constructed for prophage genes CDR20219_1208 and CDR20291_1436 (Table S2) to confirm the presence of prophage within C. difficile biofilms. PCR was carried out using Fusion High-Fidelity DNA polymerase (NEB, USA) following the manufacturer’s protocol. Samples were heated to 95 °C for 5 mins followed by 35 cycles of: 95 °C for 30 seconds, 51 °C for 30 seconds and 72 °C for 30 seconds, after which samples were heated to 72 °C for 10 mins.

PCR amplicons used in plasmid cloning were generated using Fusion, High Fidelity DNA polymerase (New England Biolabs) and purified using Qiagen PCR purification or Qiagen Gel extraction kits. For diagnostic PCR amplification, GoTaq (Promega) DNA master mix was used. All constructed plasmids were sequenced to verify authenticity (Biobasic, Canada). For parasite genomic DNA extraction, total cell pellets were first treated with 0.15% saponin in PBS for 10 min, then washed with PBS before DNA was extracted using a DNeasy Blood & Tissue Kit (Qiagen).