Pcrbio hifi polymerase

The prokaryotic primers 515F (GTGCCAGCMGCCGCGGTAA) and 806R (GGACTACHVGGGTWTCTAAT), along with Illumina Nextera overhang adapters, were used to amplify the V4 region of the bacterial 16S rRNA region (Caporaso et al., 2011 (link)). For the first PCR run, thermocycler conditions were 95°C for 2 min, 33 cycles of 95°C for 15 s, 55°C for 15 s, 68°C for 40 s; and final elongation at 68°C for 4 min (SimpliAmp Thermal Cycler, Applied Biosystems, California, USA). Each PCR reaction of 25 μL consisted of 5xPCRBIO HiFi Buffer (5 μL) m (PCR Biosystems, United Kingdom), 2 ng of DNA template, 0.25 unit of PCRBIO HiFi Polymerase (PCR Biosystems, UK), 0.5 mM of forward and reverse primers, 0.5 μL of bovine serum, and 16.25 μL of H20. A second PCR (PCR2) run was performed to add unique index combinations (i7 and i5) and adaptors. For PCR2, thermocycler conditions were 98°C for 1 min, 13 cycles of 98°C for 10 s, 55°C for 20 s, 68°C for 40 s, and a final elongation at 68°C for 5 min. Subsequently, the amplicon product was cleaned using HighPrep™ magnetic beads (MagBio Genomics Inc. Gaithersburg, USA) according to the manufacturer’s instructions. Finally, amplicon libraries were pooled in equimolar concentrations and sequenced using the Illumina MiSeq platform at Aarhus University.

The archaeal primers used were 340 F (CCCTAHGGGGYGCASCA^{54 (link)}) and 915 R (GWGCYCCCCCGYCAATTC^{54 (link)}), which amplify a DNA fragment of ~ 560 bp of the 16 S rRNA gene (variable regions 3–5). 25 µL PCR reactions in duplicate were run for each sample using 1X Platinum® High fidelity buffer, 100 µM of each dNTP, 1.5 mM MgSO₄, 1 U Platinum® Taq DNA Polymerase High Fidelity (Thermo Fisher Scientific, USA), 400 nM of each V3-V5 primer mix, and 10 ng template DNA. PCR conditions were 95 °C, for 2 min followed by 35 cycles of {95 °C, for 20 s, 50 °C for 30 s, 72 °C for 60 s} and a final step of elongation at 72 °C for 5 min. PCR products were purified using Agencourt AmpureXP (Beckman Coulter, USA) with a ratio of 0.8 bead solution/PCR solution. Illumina adapters and barcodes were added with a second PCR. 2 µL purified PCR product from above was used as template for a 25 µL PCR reaction containing 1X PCRBIO Reaction buffer, PCRBIO HiFi Polymerase (PCR Biosystems, United Kingdom). PCR conditions were 95 °C, for 2 min, 8 cycles of {95 °C, for 20 s, 55 °C for 30 s, 72 °C for 60 s} and a final step of elongation at 72 °C for 5 min.

Our molecular cloning toolbox comprised an assortment of commercial products and services. Synthesis of modified and unmodified primers and sequencing of DNA samples were performed by Eurofins Genomics (Ebersberg, Germany) and Microsynth Seqlab (Göttingen, Germany), respectively. PCR reactions were conducted using either PCRBIO HiFi polymerase (PCR Biosystems, London, UK) or PfuTurbo Cx HotStart DNA polymerase (Agilent), if encountering uracil stalling. Clean-up of DNA fragments from agarose gels or PCR reactions was done using the innuPREP DOUBLEpure kit (Analytik Jena, Jena, Germany). Restriction enzymes for cloning and control digestions and T4 DNA ligase for ligation of blunt or sticky ends were purchased from Thermo Fisher. Plasmid DNA was isolated using the QIAprep^TM Spin Miniprep kit (Qiagen, Hilden, Germany). Unless indicated otherwise, all reactions using commercial products were performed according to the manufacturers’ guidelines.

Common molecular biology procedures including plasmid DNA transformation, restriction and amplification were performed as previously described by Sambrook et al. [31 ]. All modification enzymes and restriction endonucleases were used according to the manufacturer’s instructions (New England Biolabs, Ipswich, MA, USA). Mini- and midi-scale plasmid preparations were obtained using the NucleoSpin plasmid and the NucleoBond Xtra Midi plasmid purification kits, respectively (Macherey-Nagel GmbH & Co., KG, Düren, Germany). Following PCR amplification, DNA fragments were purified with the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel GmbH & Co, KG, Düren, Germany). PCR reaction was carried out in an Applied Biosystems SimpliAmp thermal cycler (Thermo Scientific, Waltham, MA, USA) using PCRBIO Classic Taq and PCRBIO HiFi polymerase (PCR Biosystems Ltd., London, United Kingdom) on C. jejuni NCTC 11168 genomic DNA template. All plasmids and primers used in this work are listed in Table S1.

Single-cell libraries that were included in the original screening which matched clones A or clone B were identified to be used for verifying selected mutants (summarized in Additional file 6 Table S4). An additional clone (Clone C (TCRα: CAAHSPYSGNTPLVF, TCRβ: CASSSGTAYNEQFF) was used as a control to determine whether mutations could be found as artifacts in unrelated T cells. Primers were designed to span both the gSNV, and the putative variants and samples were subjected to 35 cycles of PCR (Tm: 67 °C) (PCRBIO HiFi Polymerase, PCR Biosystems) yielding approximately 1000 bp amplicons (Additional file 6 Table S4). Additionally, primers contained handles (similar to those used for TCR screening) so that secondary amplification cycles could be used to index samples if necessary. Amplified samples were analyzed by gel electrophoresis, and bands were excised for DNA isolation (Nucleospin Gel Clean Up, Techtum). Gel-purified DNA samples were sent for Sanger sequencing (KI Gene Facility, CMM, Karolinska Institute) using primers specific for the universal handle incorporated onto each Forward primer (Additional file 6 Table S4). Sanger sequencing results were analyzed visually using the software package 4peaks and are summarized in Additional file 6 Table S4.