PFGE analysis of K. pneumoniae isolates was performed using XbaI as the restriction endonuclease, as previously described34 (link),35 (link). Salmonella enterica serovar Braenderup H9812 digested with XbaI was used as the reference marker. The PFGE results were analyzed using InfoQuest software version 4.5 (Bio-Rad Laboratories, Hercules, CA, USA). S1 nuclease-PFGE was performed to determine the plasmid profiles. The genomic location of mcr-8 was indicated by Southern hybridization using a digoxigenin-labeled mcr-8 probe according to the manufacturer’s instructions for the DIG-High Prime DNA Labeling and Detection Starter Kit II (Roche Diagnostics, Basel, Switzerland).
Infoquest software version 4
Manufactured by Bio-Rad
Sourced in United States
InfoQuest software version 4.5 is a data analysis tool designed for life science researchers. It provides functionality for organizing, visualizing, and interpreting experimental data.
Automatically generated - may contain errors
Lab products found in correlation
Dig high prime dna labeling and detection starter kit 2, by Roche (1 mentions)
Chef mapper pfge system, by Bio-Rad (1 mentions)
S1 nuclease, by Takara Bio (1 mentions)
Chef mapper system, by Bio-Rad (1 mentions)
Xbai, by Takara Bio (1 mentions)
Chef mapper electrophoresis system, by Bio-Rad (1 mentions)
4 protocols using infoquest software version 4
1
PFGE Analysis of K. pneumoniae Isolates
2
Plasmid Validation via S1-PFGE
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S1-pulsed-field gel electrophoresis (PFGE) was employed to validate both the size and number of plasmids present in the transconjugant and clinical strains. To achieve this, bacterial whole-cell DNA from the clinical isolates and their transconjugants was embedded in agarose plugs and subjected to digestion with S1 nuclease (Takara, Tokyo, Japan). As a reference marker, Salmonella enterica serovar Braenderup H9812, digested with XbaI, was utilised. The DNA fragments were separated using the CHEF-Mapper PFGE system (Bio-Rad) under the following conditions: 14 °C, 6 V/cm, and a 120° pulse angle for 16 h, with the initial and final pulses lasting 2.16 and 63.8 s, respectively. Subsequently, the PFGE results were analysed using InfoQuest software version 4.5 (Bio-Rad Laboratories, Hercules, CA, USA).
3
Genetic Relatedness Analysis of K. pneumoniae
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Pulsed-field gel electrophoresis (CHEF-MAP-PER System, Bio-Rad Laboratories, Hercules, CA, USA) was used to assess the genetic relatedness between the test isolates, as previously described.7 (link) Briefly, K. pneumoniae isolates from the patients were embedded in SeaKem® Gold Agarose gels, digested with XbaI, and subjected to PFGE. Salmonella enterica serovar Braenderup H9812 digested with XbaI was used as a reference marker. Chromosome DNA of different strains was separated at 6 V/cm for 19.5 hrs at 14°C, with pulse times of 2.16–63.8 s and an included angle of 120°. PFGE profiles were analyzed by InfoQuest software version 4.5 (Bio-Rad Laboratories, Hercules, CA, USA). MLST was performed on all isolates to investigate the scale of clonal dissemination of the test strains by multiplex polymerase chain reaction (PCR) assay.11 (link)
4
Pulsed-field Gel Electrophoresis of Bacterial Isolates
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PFGE of 103 isolates was performed following the PulseNet protocol with modification [47 (link)]. Briefly, 200 μl of the overnight culture of each isolate was added to agarose for plug preparation. Plugs were lysed at 54 °C for 2 h, and the obtained DNA was digested with 50 U of XbaI (TaKaRa, Dalian, China) at 37 °C for 4 h. The restriction fragments were separated in a 0.5 × Tris-borate-EDTA buffer at 14 °C for 18 h using a Chef Mapper electrophoresis system (Bio-Rad Laboratories, Hercules, CA, USA) with switch times of 2.16 s and 63.8 s. The gels were stained with GoldenView and visualized under UV transillumination. Salmonella Braenderup H9812 was used as the standard molecular marker. Images were analyzed using the InfoQuest software version 4.5 (Bio-Rad Laboratories). The PFGE patterns were analyzed according to the Dice similarity coefficient method using the BioNumerics software (version 7.6; Applied Maths, Kortrijk, Belgium). The unweighted pair group method with arithmetic mean (UPGMA) was selected to construct a dendrogram with a position tolerance of 1.5% and optimization of 1.5%.
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