Phusion hi fidelity dna polymerase

Oligonucleotides used in this study are listed in Supplementary Data 2. gBlocks designed for capturing of GPA BGCs are listed in Supplementary Table 1. Oligonucleotides and gBlocks were ordered from Integrative DNA Technologies (Coralville, IA, USA) and Sanger and genome sequencing were performed at the MOBIX Lab Central Facility (McMaster University). PCR reactions were performed using Dream Taq Green PCR Master Mix (2×) and Phusion Hi-Fidelity DNA polymerase (Thermo Fisher Scientific). Plasmids were purified using the GeneJet Plasmid Miniprep Kit (Thermo Fisher Scientific) except for pGP1529, pGP1416, and pGP6738 which were purified using the alkaline lysis method. Restriction enzymes were purchased from Thermo Fisher Scientific and T4 DNA ligase was purchased from New England Biolabs.

A 500 bp region of the 16S rRNA gene (covering V1–V3) was PCR-amplified from 5 ng of extracted DNA sample using primers comprising universal 16S primers 27F (Frank et al., 2008 (link)) and 519R (Lane et al., 1985 (link)) along with Roche GS-FLX Titanium Series adapter sequences (A & B) for 454 pyrosequencing. The forward primers incorporate 12 base unique barcode sequences (5′-CCATCTCATCCCTGCGTGTCTCCGACTCAG-NNNNNNNNNNNN-AGAGTTTGATYMTGGCTCAG-3′) to enable pooling of samples in the same sequencing run. The appropriate barcoded A-27F and the B-519R (5′-CCTATCCCCTGTGTGCCTTGGCAGTCTCAG-GWATTACCGCGGCKGCTG-3′) primers were used in PCRs with PHUSION Hi-Fidelity DNA polymerase (Thermo Scientific). For the reaction conditions, there was an initial denaturation step of 30 s at 98°C followed by 25 cycles of 98°C for 10 s, 50°C for 30 s, 72°C for 1 min, and a final extension of 72°C for 10 min. PCR amplicons were initially checked using agarose gel electrophoresis and purified using Ampure magnetic beads according to the manufacturer's instructions. Amplicon quantification, QC, pooling, and unidirectional sequencing of the samples was performed using the Lib-L kit and the Roche 454 GS-FLX + Titanium series sequencer by the DNA sequencing facility (Dr. Shilo Dickens), Department of Biochemistry, Cambridge University, Cambridge, UK.