Q5 high fidelity pcr kit

A minigene construct was prepared by isolating the entire HTT exon 49–50 region of interest (Supplementary Table 1) from HEK293T genomic DNA. We used the Q5® High-Fidelity PCR Kit (New England Biolabs, E0555S) with the following conditions: initial denaturation 98 °C (3 min), 35 cycles of 98 °C (10 s), 64 °C (30 s), 72 °C (60 s), final extension 72 °C (2 min). This PCR fragment was TOPO cloned into pcDNA™3.1/V5-His backbone (Invitrogen, V81020). We used in vivo assembly cloning^{61 (link),62 (link)} for site-directed mutagenesis to modify the nucleotide 1 bp upstream of the exon 49 splice junction to each of the alternative nucleotides (Supplementary Table 1). The PCR for cloning was with UltraRun® LongRange PCR Kit (QIAGEN, 206442) with the following conditions: initial denaturation 93 °C (3 min), 18 cycles of 93 °C (30 s), 60 °C (15 s), 68 °C (3 min 35 s), final extension 72 °C (10 min). The amplicons were treated with DpnI restriction enzyme to remove the plasmid template and transformed into XL10 gold competent cells prepared by Mix and Go! transformation kit (Zymo Research, T3001). The sequence of the isolated plasmids was confirmed using nanopore sequencing (Plasmidsaurus, SNPsaurus LLC). Confirmed plasmids were transfected into HEK293T cells with lipofectamine 3000 (Invitrogen, L3000001) following the manufacturer’s protocol.

Desired mutations were introduced to N-terminally c-Myc tagged human CTR in pENTER11 (Invitrogen) via the Q5® High-Fidelity PCR Kit (New England Biolabs), then LR recombination reactions were conducted to transfer mutated and wild-type (WT) receptor into the pEF5/FRT/V5-DEST destination vector using Gateway Technology (Invitrogen). The oligonucleotides for mutagenesis were purchased from Gene-Works (Thebarton, SA, Australia) and mutants were confirmed by automated-sequencing.

Av. paragallinarum was isolated from the nasal cavity on day 4 or 6 post infection using a wet swab and was subsequently transported to the laboratory in ice-cold D-PBS (Sigma-Aldrich, St. Louis, MO, USA, Cat. no. D5773-50L).
Genomic DNA was extracted using a commercial QIAamp MinElute Virus Spin Kit (Qiagen, Hilden, Germany, Cat. no. 57704) according to the manufacturer’s instructions. The target region for PCR included the hypervariable region of the hmtp210 gene [25 (link)], which was amplified with the following primers: forward primer, GGC GAT TTA ACA CGG GAG TC, and reverse primer, TCAT ACC AGA TAA ACG GAT ACCT. A Q5 High-Fidelity PCR Kit (New England Bio Labs, Cat. no. E0555) containing pre-mixed dNTP, Taq polymerase, MgCl₂ and buffer at optimum concentrations was used for PCR. The thermal cycling steps used for amplification were as follows: 98 °C for 30 s; 35 cycles at 98 °C for 10 s, 58.6 °C for 15 s, and 72 °C for 20 s; and a final step at 72 °C for 2 min. The PCR products, 194 bp in length, were subjected to agarose gel electrophoresis on 2% agarose gels, after which they were visualized and imaged under ultraviolet light (Azure Biosystems, Dublin, CA, USA).