PFGE of XbaI-digested (TaKaRa, Japan) genomic DNA of blaNDM-1-positive E. cloacae and reference marker Salmonella serotype Braenderup strain (H9812) was performed using a contour-clamped homogeneous electric field (CHEF)-Mapper XA PFGE system (Bio-Rad, USA) for 22 h at 6 V/cm and 14°C, with a pulse angle of 120° and pulse times from 5 to 35 s. Comparison of the PFGE patterns was performed with InfoQuest FP software version 4.5 (Bio-Rad Laboratories, USA) using the Dice similarity coefficient. Clusters were defined as DNA patterns sharing >85% similarity. MLST was carried out as described previously[18 (link)], the database available at http://pubmlst.org/ecloacae was used for assigning STs.
Infoquest fp software version 4
Manufactured by Bio-Rad
Sourced in United States
InfoQuest FP software version 4.5 is a data analysis tool designed for processing and analyzing fluorescence polarization (FP) data. The software provides a user-friendly interface for importing, visualizing, and analyzing FP data generated from various experimental setups.
Automatically generated - may contain errors
Lab products found in correlation
Chef mapper system, by Bio-Rad (2 mentions)
Chef mapper xa pfge system, by Bio-Rad (1 mentions)
Xbai, by Takara Bio (1 mentions)
Chef mapper apparatus, by Bio-Rad (1 mentions)
Chef mapper xa pulsed field gel electrophoresis system, by Bio-Rad (1 mentions)
Lambda dna ladder, by Bio-Rad (1 mentions)
Pulsed field certified agarose, by Bio-Rad (1 mentions)
Xbai restriction enzyme, by New England Biolabs (1 mentions)
Tianamp bacteria dna kit, by Tiangen Biotech (1 mentions)
Chef dr 3, by Bio-Rad (1 mentions)
8 protocols using infoquest fp software version 4
1
PFGE Typing of Carbapenem-Resistant E. cloacae
2
PFGE Analysis of CTX-M-producing E. coli
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Pulsed-field gel electrophoresis (PFGE ) was performed on CTX-M–producing E. coli isolates by digesting the genomic DNA using the XbaI (Takara Bio Inc., Shiga, Japan) enzyme according to the standard protocol of the Center for Disease Control and Prevention and CHEF MAPPER apparatus (Bio-Rad Laboratories, Hercules, CA), as previously described (Liu et al., 2007 (link)). Gel images were analyzed using InfoQuest FP software, version 4.5 (Bio-Rad). The dice coefficient was used to calculate similarity, and the similarity matrix was expressed graphically by an unweighted average linkage.
3
PFGE Analysis of Salmonella Serovars
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The chromosomal DNA of S. Derby, S. Indiana, S. Typhimurium, and S. Enteritidis strains were digested with restriction enzyme XbaI and subjected to PFGE according to the Pulse Net Standardized Laboratory Protocol (U.S. Centers for Disease Control and Prevention, Atlanta, GA) using the CHEF Mapper XA Pulsed Field Gel Electrophoresis System (Bio-Rad). Electrophoresis parameters: 6.0 V/cm, 120°angle, initial and final switch times of 5 and 40s, respectively; run time was 18.5 h at 14 °C. Cluster analysis of PFGE types was performed according to the Dice coefficient method [33] (link) using the InfoQuest FP Software/Version 4.5 (Bio-Rad).
4
Genetic Relationships of Tetracycline-Resistant E. faecalis
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The genetic relationships among tetracycline-resistant E. faecalis harboring at least one or more tet genes were evaluated based on PFGE carried out with the CHEF-Mapper system (Bio-Rad) [37 (link)]. Genomic DNA was digested with 20 U SmaI (Takara Bio, Kyoto, Japan) and separated on 1.0 % pulsed-field certified agarose (Bio-Rad). Running conditions were 6.0 V/cm at 14 °C for 20 h with pulse times ramped from 1 to 20 s in 0.5 × TBE buffer. A lambda DNA ladder (Bio-Rad) was used as the size marker. A cluster analysis of the PFGE results was conducted to determine relatedness of tetracycline-resistant isolates using the InfoQuest FP Software version 4.5 (Bio-Rad) with the Dice co-efficient and the unweighted pair group method with arithmetic averages. Optimization settings for the dendrogram were 0.5 % with a band tolerance of 0.1 %.
5
Genomic Fingerprinting of E. coli Isolates
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Pulsed-field gel electrophoresis (PFGE) was performed with the XbaI restriction enzyme (New England Biolabs, Beverly, MA, USA) as described previously.34 (link) PFGE profiles were analyzed with InfoQuest FP software version 4.5 (Bio-Rad Laboratories Inc., Hercules, CA, USA). In order to establish the epidemiological relationship among the isolates from the electrophoretic patterns, the Dice coefficient was used and clustering was based on the unweighted pair group method with arithmetic mean with a 1% tolerance in band position differences. The isolates were considered to belong to the same epidemiological group when the PFGE-XbaI profiles showed ≥80% of homology, adapting the criteria described by Tenover et al.35 (link) Multi-locus sequence typing (MLST) was carried out by amplification and sequencing of the 7 E. coli housekeeping genes as described previously.36 (link) The database available at http://mlst.warwick.ac.uk/mlst/dbs/Ecoli/ was used for assigning sequence types (STs) and clonal complexes (CCs). Classification of isolates into E. coli phylogenetic groups was done using a previously described triplex PCR-based protocol.37 (link)
6
Clonal Relatedness Analysis of K. pneumoniae
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The clonal relatedness of all K. pneumoniae clinical isolates was determined by enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) using primers, ERIC-1 (5’-ATGTAAGCTCCTGGGGATTCAC-3’) and ERIC-2 (5’-AAGTAAGTGACTGGGGTGAGCG-3’) as those previously described (Versalovic et al., 1991 (link)). The genomic DNA of all isolates was extracted using a TIANamp bacteria DNA kit (Tiangen Biotech, Beijing, China) according to the manufacturer’s protocol. The PCR amplification was performed with slight modification as follows: initial denaturation at 94°C for 5min, followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 38°C for 1 min, and extension at 72°C for 3 min, with a final extension at 72°C for 10 min. ERIC-PCR patterns were compared by InfoQuest™FP Software, version 4.5 (Bio-Rad, Hercules, USA) using the Dice coefficient. The dendrogram was generated by the unweighted pair group method with arithmetic means (UPGMA) using 1.0% optimization and 1.0% band position tolerance. Percentage similarities were used to assess the relatedness of the clones (Widerström et al., 2007 (link)).
7
PFGE Genotyping of Campylobacter Isolates
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Representative Campylobacter isolates containing RE-cmeABC were genotyped by PFGE, which was conducted with a CHEF-DR III apparatus (Bio-Rad Laboratories, Hercules, CA) in accordance with the protocol for Campylobacter (43 (link)). The DNA of Campylobacter was digested with SmaI, and the DNA of Salmonella H9812 digested with XbaI was used as the reference marker. The results were analyzed with the InfoQuest FP software version 4.5 (Bio-Rad Laboratories).
8
Molecular Surveillance of Salmonella enterica Serovar Indiana
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Chromosomal DNA of 104 Salmonella enterica serovar Indiana isolates carrying the int1, blaTEM, floR, tetA, strA, and aac(6’)-Ib-cr genes were digested with the restriction enzyme XbaI and then subjected to PFGE analysis according to the Pulse Net Standardized Laboratory Protocol (U.S. Centers for Disease Control and Prevention, Atlanta, GA) using the CHEF MAPPER System (Bio-Rad Laboratories, Hercules, CA). The gels were run at 6.0 V/cm with an initial and final switch time of 2.16 s and 54.17 s at an angle of 120 degrees and 14°C for 18 h. The Salmonellaser. Braenderup H9812 standard served as size markers. Cluster analysis of pulsotypes was carried out using Dice's coefficient in UPGMA with InfoQuest FP Software/Version 4.5 (Bio-Rad). [12]
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