Mytaq reaction buffer

PCR analysis was performed using the primers listed in Table 1. Five multiplex PCRs were performed with different primer mix and were defined as multiplex 1–5 for primer mix of bla_KPC, KPC, bla_OXA-48-like, RepA genes; bla_VIM type, VIM2, NDM-1 genes; bla_NDM-1, bla_IMP variant genes (except, IMP-3, IMP-16, IMP-27, IMP-31, IMP-34, and IMP-35) with bla_OXA-23-like; OXA-58-like, VIM1, NDM genes; and OXA-23-like, OXA-24-like, and OXA-51-like genes, respectively.
PCR amplification for carbapenemase-encoding genes was performed in a final volume of 25 μl. Reaction mixtures contained 5 μl of 5 × MyTaq Reaction Buffer (Bioline, UK), 2.5 U of MyTaq™ DNA polymerase (Bioline, UK), and forward and reverse primers are used at a final concentration of 0.2 μM each and 3 μl of extracted DNA (10–100 ng).
The amplification procedure was as follows: predenaturation at 95°C for 10 min; 40 cycles of denaturation at 95°C for 15 s, various annealing conditions (Table 1) for 15 s, and extension at 72°C for 10 s. PCR was used on a thermal cycler (MyCycler™, Bio-Rad, Germany). Electrophoresis on 1% agarose gel was used to analyze the amplicons and visualize them under UV light.

DNA was extracted from leaf tissue samples using a modified CTAB (cetyl trimethylammonium bromide) protocol (Wolff, 1996 ). Partial mitochondrial genome assemblies (constructed from Illumina, San Diego, CA, USA; paired-end reads; N Levsen and K Wolff, unpublished) of eight geographically disparate European P. lanceolata individuals were used to design PCR and sequencing primers (Supplementary Table S1) for fragments of two mitochondrial genes (atp6 and rps12). PCR amplification was conducted in a 20 μl volume consisting of 4.2 μl of 5 × MyTaq Reaction Buffer (Bioline, London, UK), 0.5 μl (10 pM) of each primer, 13.4 μl H₂O, 0.4 μl MyTaq DNA polymerase and 1 μl of template DNA. The temperature cycling protocol was as follows: 95 °C initial denaturation for 3 min; 35 cycles at 95 °C for 15 s, 52 °C for 15 s and 72 °C for 30 s; a final extension step at 72 °C for 5 min. Sanger sequencing of both DNA strands was performed at the Edinburgh Genomics facility (Edinburgh, UK). The sequence reads were quality trimmed and aligned to the Mimulus guttatus mitochondrial reference sequence (GenBank: JN098455.1; Mower et al., 2012 (link)) in CLC Genomics Workbench v8.0.2 (CLCbio, Arhus, Denmark). The annotated M. guttatus sequence provided a published guide to determining site position and category (for example, synonymous).

The chromosomal DNA of each isolate was purified from a 2 mL overnight culture grown in LB at 37 °C, incubated in a shaker at 120 rpm, using the ZymoBIOMICS DNA Miniprep Kit (Zymo Research Corp., Irvine, CA, USA), according to the manufacturer instructions. PCR amplification of the 16S rRNA gene was carried out using the 27F and 1492R oligonucleotide primers [46 (link)], at an annealing temperature of 55 °C. Each 25 μL reaction was composed of 1 × x MyTaq Reaction Buffer (Bioline, BIOPORTUGAL, Lda., Lisboa, Portugal), 0.2 μM of each primer (STAB Vida, Lisboa, Portugal), 1.25 U of MyTaq DNA Polymerase (Bioline, BIOPORTUGAL, Lda., Lisboa, Portugal), and 2.5 µL of DNA template. Sequencing of the amplified products was carried out using the primer 27F [46 (link)] by STABVIDA (Caparica, Portugal). The sequencing data were analyzed by BLASTn against a database of non-redundant bacterial sequences from NCBI (https://www.ncbi.nlm.nih.gov (accessed on 3 May 2020)).