Gotaq enzyme

Two microliters of purified pCRISPR from the integration reactions was used for adapter PCRs. Fifty microliter PCR reactions were performed with primers listed in Table S1 and Promega GoTaq enzyme. Thirty-four cycles of 98 °C for 30 s, 58 °C for 30 s, and 72 °C for 15 s were used for the PCR with initial denaturation at 98 °C for 5 min and final extension at 72 °C for 2 min. PCR products were analyzed on a 2% agarose gel prestained with SYBR Safe. Forty microliters of the PCR was used for gel extraction. Bands corresponding to the expected size of 175 bp were excised and purified using Promega Wizard gel-extraction kit.
A total of 2.5 μl of purified adapter PCR products were used for an additional PCR to add indices to samples for multiplexed MiSeq sample submission. Eight cycles of 98 °C for 30 s, 67 °C for 30 s, and 72 °C for 10 s were performed for the PCR with initial denaturation at 98 °C for 30 s and final extension at 72 °C for 2 min. PCR products were analyzed on a 2% agarose gel prestained with SYBR Safe. Equal volumes of the final PCR reactions were mixed, and gel purified with the Promega Wizard gel-extraction kit. Sample quality was checked using Qubit and Agilent Bioanalyzer. Samples were sequenced using MiSeq with 2 × 75 cycles at the Iowa State University DNA Facility.

Briefly, mid-log cultures of donor (HA2pEho and HA7pKpn) and recipient (E. coli J53) strains were mixed in LB broth (Laboratorios Britania S.A., Argentina). The mating culture was then incubated overnight at 37°C using the drop plate method. Four replicas were used in each conjugation: only LB, LB with the addition of meropenem (8 µg/ml), sodium azide (80 µg/ml), or a combination of both. To verify that colonies growing on both antibiotics were transconjugant E. coli J53, they were grown on EMB agar (Laboratorios Britania S.A., Argentina). bla_KPC-2 PCR was carried out with the primers KPC-F: CCGTCAGTTCTGCTGTC and KPC-R: CGTTGTCATCCTCGTTAG (Ramírez et al., 2013 (link)) using GoTaq^® enzyme (Promega, Madison, WI).

2 μL of purified pCRISPR from the integration reactions was used for adaptor PCRs. 50 μl PCR reactions were performed with primers listed in Table S1 and Promega GoTaq enzyme. 34 cycles of 98 °C for 30 s, 58 °C for 30 s and 72 °C for 15 s were used for the PCR with initial denaturation at 98°C for 5 min and final extension at 72°C for 2 min. PCR products were analyzed on a 2% agarose gel pre-stained with SYBR Safe. 40 μL of the PCR was used for gel extraction. Bands corresponding to the expected size of 175 bp were excised and purified using Promega Wizard gel-extraction kit.
2.5 μL of purified adaptor PCR products were used for an additional PCR to add indices to samples for multiplexed MiSeq sample submission. 8 cycles of 98 °C for 30 s, 67 °C for 30 s and 72 °C for 10 s were performed for the PCR with initial denaturation at 98 °C for 30 s and final extension at 72 °C for 2 min. PCR products were analyzed on a 2% agarose gel pre-stained with SYBR Safe. Equal volumes of the final PCR reactions were mixed, and gel purified with the Promega Wizard gel-extraction kit. Sample quality was checked using Qubit and Agilent Bioanalyzer. Samples were sequenced using MiSeq with 2 × 75 cycles at the Iowa State University DNA Facility.

Total RNA was extracted using Geneaid Minikit (Geneaid). After digestion with DNase, 1 μg of RNA was reverse-transcribed using M-MLV reverse transcriptase (Promega) and random hexamers (Promega) and used as template for conventional PCR analyses using GoTaq enzyme (Promega), before analysis on agarose or acrylamide gels and quantification by densitometry using Image Lab Software (Biorad).⁵⁵ (link) Real-time quantitative PCRs (qPCR) were performed using the SYBR Green I Master and the LightCycler 480 System (Roche). All primers used are listed in the Table S7.

Total RNA was extracted from cells seeded in 60 mm Petri dishes with the RNeasy Mini Kit (Qiagen, Venlo, Netherlands). RT-PCR was performed using M-MLV Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) using 500 ng of total RNA and the GoTaq® enzyme (Promega, Madison, WI, USA) according to manufacturer’s recommended procedures [13 (link)]. The primers used for RT-PCR were GFP F: 5′-AAGGCTACGTCCAGGAGCGC-3’, R: 5′-CTTGTGCCCCAGGATGTTGC-3’; NS1: F 5′-AGAGGACCATCTCTGAGATC-3’, R 5′-GGCCTTATCTCCATTCCATACC-3’; NS3: F 5′-ATGCACACTGGCTTGAAGC-3’, R 5′-CAGATGCAACCTGATAGGC-3’; RNA PolII: F 5′-GCACCACGTCCAATG-3’, R 5′-GTGCGGCTGCTTCCA-3’; GAPDH: F 5′-GGGAGCCAAAAGGGTCATCA-3’, R 5′-TGATGGCATGGACTGTGGTC-3’. RT-PCR products were visualized by agarose gel electrophoresis. qPCR was performed using the GoTaq^® qPCR Master Mix (Promega). All steps were conducted according to the manufacturer’s instructions. The qPCR data were analyzed using the ΔΔCt method and results were normalized to GAPDH, which was used as an internal control.