The XbaI-digested genomic DNA’s pulsed field gel electrophoresis (PFGE) pattern and the multi-locus sequence type of the isolates were established as described earlier.6 (link),7 (link) The Kuwaiti K. pneumoniae isolates were also compared by repetitive element sequence-based polymerase chain reaction (rep-PCR; DiversiLab; bioMerieux, Marcy l’Etoile France) using the Klebsiella kit, according to the manufacturer’s recommendation. Resistance genes (blaTEM, blaCTX-M, blaSHV, blaPER, blaAmpC, blaNDM, blaOXA-48-like, blaKPC, blaVIM, blaIMP, armA, rmtA, rmtB, rmtC, rmtD, rmtE, rmtF, qnrS, qepA, aac6-1b-cr, mcr-1, mcr-2) were detected as described.4 (link),7 (link),11 (link),12 The specific alleles of beta-lactamase genes were determined by direct sequencing of the respective amplicons performed with the BigDye Cycle Terminator V.3.1 (Thermo Fisher Scientific, Waltham, MA, USA) using the 3130X Genetic Analyzer (Thermo Fisher Scientific).
Bigdye cycle terminator v 3
Manufactured by Thermo Fisher Scientific
Sourced in United States
BigDye Cycle Terminator V.3.1 is a reagent kit used in DNA sequencing. It contains dye-labeled terminators that enable the detection and identification of DNA sequences.
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Lab products found in correlation
2 protocols using bigdye cycle terminator v 3
1
Molecular Characterization of Klebsiella pneumoniae
2
Plasmid Purification and Sequencing
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PCR amplicons were purified with Wizard ® SV Gel and PCR Clean-Up System (Pomega, USA). Plasmids were purified from single plasmid containing transconjugants or transformants using Plasmid Maxi Kit (Qiagen). Sequencing was performed with the Big Dye Cycle Terminator V.3.1 (Thermo Fisher Scientific, Waltham, MA, USA) on the 3130X Genetic Analyzer (Applied Biosystems) according to the manufacturer's instructions. The sequences obtained were analyzed using MEGA 4 and assembled with Clone Manager v9 software.
The assembled sequences were annotated using Sequin (http://www.ncbi. nlm.nih.gov/Sequin) and submitted to GenBank.
The assembled sequences were annotated using Sequin (http://www.ncbi. nlm.nih.gov/Sequin) and submitted to GenBank.
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