Velocity proof reading dna polymerase

Restriction enzymes were purchased from New England Biolabs; T4 DNA ligase and Taq DNA polymerase were from Invitrogen; Velocity proof-reading DNA polymerase was from Bioline; Q5 high fidelity Taq polymerase was from NEB. PCR amplification of DNA was achieved using the primers (AltaBioscience, University of Birmingham, UK; or Sigma Aldrich) listed in S3 Table. Reactions were cycled in a SensoQuest Lab Cycler following standard procedures [57 ]. PCR products were purified using the Illustra GFX^TM PCR DNA and Gel Band Purification Kit (GE^TM Healthcare). Small-scale plasmid DNA preparations were performed using the AccuPrep Plasmid MiniPrep DNA Extraction Kit (Bioneer) adapted from the alkaline lysis method of Birnboim and Doly [58 (link)]. DNA sequencing reactions were prepared and run on an ABI 3730 DNA analyser (Functional Genomics Facility, University of Birmingham, U.K.) following the chain termination method [59 ].

Bacterial isolates were identified to genus-, or where possible species-level. Bacterial cultures were grown in LB from single colonies and genomic DNA was extracted using a Qiagen Blood and Tissue DNeasy kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. Partial 16S rDNA fragments (~2 kb) were amplified from 10–30 ng of genomic DNA by PCR using the forward primer 27 F (AGA GTT TGA TCC TGG CTC AG) and reverse primer 1522 R (AAG GAG GTG ATC CAG CCG CA). PCR was carried out using Velocity proofreading DNA polymerase (Bioline) according to manufacturer’s instructions. The PCR cycling conditions were a denaturation of 94 °C for 2 min followed by 30 cycles of 94 °C for 30 sec, 58 °C for 30 sec and 72 °C for 1 min, then a final extension of 72 °C for 5 min. PCR products were purified using a GeneJET PCR purification kit (Thermo Fisher) and were sequenced using both the forward and reverse primers via capillary sequencing on an ABI3730 machine (Applied BioSystems). Raw sequence reads viewed in SnapGene version 1.4 and were trimmed and refined by eye from the peak trace where necessary. Where possible, forward and reverse sequences were aligned and combined to generate a single consensus sequence. Sequences were analysed by BLASTN^{95 (link)} and the closest matches recorded.