Amplification of blaNDM-1 producers: Plasmid DNA was extracted for selected phenotypically carbapenems resistant isolates using GeneJET Plasmid Miniprep
Kit by Thermo Fisher Scientific Lithuania, according to the manufacturer's instructions.
The buccal cells donated by the subjects were collected in Resuspension Solution (GenElute Mammalian Genomic DNA Miniprep
Kit, Sigma, Germany) with the use of sterile foam-tipped applicators (Puritan, USA).
Chemicals, Bacterial strains, Plasmid and Media: Chemicals; Kanamycin (Catalog number: 11815024) Thermo Fisher Scientific, Thermo Scientific GeneJET plasmid miniprep
kit, Gene ruler (SM0311 1Kb) Fermentas Inc, Thermo Scientific GeneJET Gel Extraction Kit.
Se colecto el micelio mediante centrifugacion y se extrajo el ADN con el Kit AxyPrep Multisource Genomic Miniprep
(Axygen, USA) usando el protocolo del fabricante.
The PcDNA-3.1 plasmid was purified using a plasmid DNA miniprep
system (Viogene, USA) from the bacterial culture containing the plasmids stored in glycerol stock according to the supplier's instructions.
Approximately 3ml of blood was collected in a Vacutainer Potassium-salt EDTA tube based on the protocol from the ReliaPrep[TM] Blood gDNA Miniprep
System (Promega, USA, Cat# A5081).
The gDNA extraction was performed using the Plant ZR/Seed Zymo Research Miniprep
DNA kit (Zymo Research, Irvine, United States of America) following the manufacturer's instructions.
PCR product was purified through QIAquick Spin Miniprep
kit (QIAGEN, Valencia, California, USA).
We extracted DNA from soil by using the ZR Soil Microbe DNA Miniprep
Kit (Zymo Research, Irvine, CA, USA).
The DNA pEGFP-N1 plasmid was amplified in Escherichia coli, extracted using the QIAprep Spin Miniprep
. The purity of the plasmid was checked with 1% agarose gel electrophoresis and the DNA was visualized with ethidium bromide.
The cultures were pooled, and genomic DNA was extracted from 100 mL of the mixed bacteria using an AxyPrep Bacterial Genomic DNA Miniprep
Kit (Axygen Scientific, Union City, CA, USA).
In the next step, DNA was extracted using a ZR-Duet[TM] DNA/RNA MiniPrep
kit (Zymo Research Corp., USA) according to the manufacturer's instructions.