Phusion taq dna polymerase

Microbial DNA was isolated from shrimp intestinal samples using the repeated bead beating plus column method, as described by Yu and Morrison [18 (link)]. The V1–V3 region of the bacterial 16S rRNA gene was PCR-amplified using the 27F forward [19 (link)] and 519R reverse [20 (link)] primer pair. PCR reactions were performed with the Phusion Taq DNA polymerase (Thermo Scientific, Waltham, MA, USA) under the following conditions: hot start (4 min, 98 °C), followed by 35 cycles of denaturation (10 s, 98 °C), annealing (30 s, 50 °C) and extension (30 s, 72 °C), then ending with a final extension period (10 min, 72 °C). PCR products were separated by agarose gel electrophoresis, and amplicons of the expected size (~500 bp) were excised for gel purification using the QiaexII Gel extraction kit (Qiagen, Hilden, Germany). For each sample, approximately 400 ng of amplified DNA were submitted to Molecular Research DNA (MRDNA, Shallowater, TX, USA) for sequencing with the Illumina MiSeq 2X300 platform to generate overlapping paired end reads.

Microbial genomic DNA was isolated from fecal samples using the repeated bead beating plus column method, as previously described [52 (link)]. The V1-V3 region of the bacterial 16S rRNA gene was PCR-amplified using the 27F forward [53 (link)] and 519R reverse [54 (link)] primer pair. PCR reactions were performed with the Phusion Taq DNA polymerase (Thermo Scientific, Waltham, MA, USA) under the following conditions: hot start (4 min, 98 °C), followed by 35 cycles of denaturation (10 s, 98 °C), annealing (30 s, 50 °C) and extension (30 s, 72 °C), then ending with a final extension period (10 min, 72 °C). PCR products were separated by agarose gel electrophoresis, and amplicons of the expected size (~ 500 bp) were excised for gel purification using the QiaexII Gel extraction kit (Qiagen, Hilden, Germany). For each sample, approximately 400 ng of amplified DNA were submitted to Molecular Research DNA (MRDNA, Shallowater, TX, USA) for sequencing with the Illumina MiSeq (2X300) platform to generate overlapping paired end reads.

Human CD99 D92H and D92Y mutants were produced by site-directed mutagenesis with hCD99 WT myc-/FLAG-tagged construct serving as template DNA. The following primers were used: c.274G>C forward: 5′-cta gttcctccggtagcttttcacatgctgaccttgcggatggc-3′, c.274G>C reverse: 5′-gccatccgcaaggtcagcatgtgaaaagctaccggaggaac tag-3′; c274G>T forward: 5′-ctagttcctccggtagcttttcatat gctgaccttgcggatggc-3′; c274G>T reverse: 5′-gccatccgcaag gtcagcatatgaaaagctaccggaggaactag-3′. Primers were phosphorylated, then added to template DNA and DNA was amplified by Phusion Taq DNA Polymerase (ThermoFisher). Methylated template DNA was then digested by DpnI enzyme and the amplified DNA was ligated. Introduction of both point mutations was confirmed by DNA sequencing (GATC Biotech).

The hypervariable accD-psaI intergenic spacer was amplified by PCR using accD F2 (Zheng et al., 2014 (link)) and psaI 75R (Small et al., 1998 (link)) primers. PCR reactions contained: 1X Phusion HF Buffer, 200 μM dNTPs each, 0.5 μM each primer, 0.2 U Phusion Taq DNA polymerase (Thermo Fischer Scientific), 30 ng genomic DNA and H₂O to a final volume of 50 μl. PCR amplification was carried out by GeneAmp PCR system 9700 (Applied Biosystems) programmed as follow: 98°C for 30 s, followed by 30 cycles of 98°C for 10 s, 66°C for 15 s, 72°C for 30 s, and then 72°C for 10 min.
The purified PCR products of about 1,000 bp were sequenced at the Polo di Innovazione Genomica, Genetica e Biologia (Perugia, Italy). The new cpDNA sequences were recorded in GenBank with accession numbers from KY606436-KY606530.

DNAZOL® reagent (Invitrogen, Carlsbad, CA, USA) was used for total DNA extraction of the tomato near‐isogenic lines, which was then employed as template for the amplification of the Sw‐5 genes (Sw‐5a, Sw‐5a^S, Sw‐5b) via polymerase chain reaction (PCR) using Phusion Taq DNA polymerase (Thermo Scientific, Waltham, MA, USA). The PCR products were recombined into pDONR207 entry vectors by BP Clonase Enzyme Mix (Invitrogen). Truncated gene versions were PCR amplified from their full‐length copies, cloned in pDONR207 vectors, and either ligated in pENTR11 (NcoI and XhoI restriction sites) or recombined again in pDONR207, as detailed in Table S1 (see Supporting Information). The pDONR207 constructs harbouring NS_M from the RI BR‐01 or RB GRAU strains were prepared previously (Hallwass et al., 2014). All entry vectors were recombined with one or more of the following destination vectors by LR Clonase Enzyme Mix (Invitrogen): pEAQ‐DEST1, pEAQ‐DEST2, pK2GW7, pK7WGF2 (Karimi et al., 2002; Peyret and Lomonossoff, 2013). A list of all primer sequences, entry and destination vectors of all constructs used in this study is shown in Table S1. All procedures followed the manufacturer's instructions and Green and Sambrook (2012).