Mgcl2

To assess KRAS exon 2 mutational status, 50 ng of template DNA was used for standard PCR amplification using 0.12 μM of each primer (forward: 5′-GGTGGAGTATTTGATAGTGTA-3′; reverse: 5′-TGGACCCTGACATACTCCCAAG-3′), 2 mM of MgCl₂ (NzyTech, Lisbon, Portugal), 0.8 mM of dNTP Mix (NzyTech, Lisbon, Portugal), and 0.15 units of NZYTaq II DNA Polymerase (NzyTech, Lisbon, Portugal) in a final reaction volume of 20 μL. Reactions were performed on a DNA Engine® thermocycler (Bio-Rad, Hercules, CA, USA) as follows: 95 °C for 5 min, followed by 30 cycles of 95 °C for 30 s, 61 °C for 30 s for tumor samples and 53 °C for 30 s for plasma samples, and 72 °C for 20 s. For plasma samples, four independent amplification reactions were performed for each sample, which were later concentrated using the SpeedVac Concentrator (Thermo Fisher Scientific, Waltham, MA, USA) and resuspended in 15 μL of DEPC-treated water.
PCR products were then direct sequenced at STAB VIDA (Setubal, Portugal), and the chromatograms analyzed using FinchTV software (Geospiza, Inc) for sequence characterization and identification of possible mutations in codon 12.

Medical grade CS 95/1000 (with a molecular weight range between 200 and 500 kDa was) purchased from Heppe Medical (Berlin, Germany), TPP was obtained from Acros Organics (Geel, Belgium), 35% hydrochloric acid, sodium hydroxide palettes, and glacial acetic acid were all acquired from VWR (Prague, Czech Republic). The following solutions were freshly prepared by using deionized water from VWR (Prague, Czech Republic): 2 M HCl, 10 M NaOH, 1%, and 2% (v/v) acetic acid. GRS Taq DNA polymerase was purchased from Sigma Aldrich Chemicals (St. Louis, MO, USA). TripleXtractor used in RNA extraction was obtained from GRISP (Porto, Portugal). DMEM-F12 and DMEM-HG were purchased from GIBCO (Waltham, MA, USA). Sodium bicarbonate was obtained from MP Biomedicals (Santa Ana, CA, USA). Agarose, MgCl₂ and GreenSafe were obtained from NZYtech (Lisbon, Portugal). All solutions were freshly prepared by using ultra-pure grade water, purified with a Milli-Q system from Millipore (Billerica, MA, USA).

Molecular characterization of the isolates based on spa-typing was performed. DNA was extracted by the boiling method and tested according to the protocol used by the EURL-AR [41 (link)]. The spa-type was determined using Ridom SeqSphere+ software v8 (Ridom GmbH, Münster, Germany).
Isolates with MIC ≥ 4 µg/mL for linezolid were tested for the presence of the cfr gene by standard PCR, using the primers described by Kehrenberg and Schwarz (2006) [26 (link)] for the amplification of a 746 bp fragment. The reactions were carried out in a total volume of 25 µL containing 1 × Gel Load Reaction buffer (NzyTech, Lisbon, Portugal), 2 mM MgCl2 (NzyTech), 400 mM dNTPs (NzyTech), 0.4 μM of each primer, 1U of NZYTaq II DNA polymerase (NzyTech), and 2 μL of DNA. Amplification was performed in a Biometra TOne Thermal Cycler (Analytik Jena, Jena, Germany) with an initial denaturation step at 94 °C for 5 min, followed by 30 cycles at 94 °C for 30 s, 54 °C for 90 s, and 72 °C for 60 s, with a final extension at 72 °C for 10 min. The PCR products were visualized, and images were collected using the UVP BioDoc-It^® Imaging System (UVP, Cambridge, UK).