Isolates of interest were analysed using pulsed-field gel electrophor esis (PFGE), as described by the Centers for Disease Control and Prevention, with the following changes: cultures were grown on BHI medium, at 37°C, aerobically overnight, and cell densities were standardised to an OD 610nm ≈1.0, using a spectrophotometer (BioDrop, UK). [9] Cells were embedded in a 1.0% SeaKem Gold agarose gel (Lonza, Switzerland) and were digested for 2 hours at 25°C using SmaI (Thermo Scientific, USA) restriction endonuclease and ApaI as a secondary enzyme (2 hours at 37°C). Digested plugs were electrophoresed through a 1.0% SeaKem Gold agarose gel (Lonza, Switzerland) in 0.5% TBE buffer (14 °C), using the Gene Navigator (Amersham Biosciences, UK) at 200 V, with a ramped switching time of 3.5 -23.5 seconds over 18 hours. The resultant gel was visualised using ethidium bromide staining, and the gel image analysed and dendrogram generated using Gel Compare II (BioNumerics v.6.6, Applied-Maths, USA), with an optimisation of 1.0 and a band tolerance of 1.5. The dendrogram was constructed using the pairwise average from the SmaI and ApaI results, with the UPGMA (unweighted pair group method with arithmetical mean) method and branch quality was calculated using the cophenetic correlation. E. faecalis ATCC 51299 was used as a control and for normalisation.
Seakem gold agarose gel
Manufactured by Lonza
Sourced in United States, Switzerland
SeaKem Gold agarose gel is a laboratory reagent used for gel electrophoresis. It is a purified agarose product designed for nucleic acid separation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Chef mapper xa system, by Bio-Rad (6 mentions)
Universal hood 2, by Bio-Rad (6 mentions)
Xbai restriction enzyme, by Takara Bio (5 mentions)
Chef dr 2 system, by Bio-Rad (4 mentions)
Positively charged nylon membrane, by Roche (2 mentions)
Xbai, by Takara Bio (2 mentions)
Midrange 1 pfg marker, by New England Biolabs (2 mentions)
Chemidoc xrs gel imaging system, by Bio-Rad (2 mentions)
Stains all, by Santa Cruz Biotechnology (2 mentions)
Chemidoc imaging system, by Bio-Rad (2 mentions)
Chef mapper pulsed field electrophoresis system, by Bio-Rad (1 mentions)
Gel doc xr imaging system, by Bio-Rad (1 mentions)
Chef mapper pfge system, by Bio-Rad (1 mentions)
Sybr gold solution, by Thermo Fisher Scientific (1 mentions)
Chef mapper system, by Bio-Rad (1 mentions)
Sybr safe dna gel stain, by Thermo Fisher Scientific (1 mentions)
Biodyne b nylon membrane, by Avantor (1 mentions)
3 end labeling kit, by Roche (1 mentions)
Las 4000 mini, by GE Healthcare (1 mentions)
Cdp star, by Roche (1 mentions)
23 protocols using seakem gold agarose gel
1
Pulsed-Field Gel Electrophoresis Protocol for Bacterial Isolate Typing
2
Plasmid Identification Using PFGE
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Plasmid-harboring bacteria were digested with S1 nuclease after being embedded in 1% SeaKem Gold Agarose gels (Lonza, Rockland, ME, USA). PFGE was performed as described above. The plasmid DNA was then transferred to positively charged nylon membranes (Roche Diagnostics GmbH Mannheim, Germany) and hybridized against DIG-labeled blaNDM-5-specific probes. Incompatibility group identification for blaNDM-5-carrying plasmids was conducted by hybridizing against blaIncX3 and blaIncFII-specific probes in the same manner.
3
Plasmid-Mediated IMI-2 Carbapenemase Detection
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The two isolates were embedded in SeaKem Gold Agarose gels (Lonza, Rockland, ME, USA) and digested with S1 nuclease. PFGE was performed as described previously. Plasmid DNA separated by S1-PFGE was transferred to positively charged nylon membranes (Roche Diagnostics GmbH, Mannheim, Germany) and hybridized against DIG-labeled bla IMI and incompatibility group-specific probes (Zhang et al., 2015) .
Multilocus sequence typing (MLST) and genetic structures surrounding bla IMI-2 E. coli RJ18 was analyzed by MLST according to the protocol described in the E. coli MLST website (http://mlst.warwick.ac.uk/ mlst/dbs/Ecoli). A PCR mapping approach was adopted to reveal the genetic context of the bla IMI-2 gene in pRJ18 using specifically designed primers (Table 1) according to strain W635 reported in Spain (GenBank accession number JN412066) (Rojo-Bezares et al., 2012) .
Multilocus sequence typing (MLST) and genetic structures surrounding bla IMI-2 E. coli RJ18 was analyzed by MLST according to the protocol described in the E. coli MLST website (http://mlst.warwick.ac.uk/ mlst/dbs/Ecoli). A PCR mapping approach was adopted to reveal the genetic context of the bla IMI-2 gene in pRJ18 using specifically designed primers (Table 1) according to strain W635 reported in Spain (GenBank accession number JN412066) (Rojo-Bezares et al., 2012) .
4
Genetic Relatedness by PFGE
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Determination of the genetic relationship between the integron positive isolates was accomplished by pulsed-field gel electrophoresis (PFGE) analysis according to the protocols and criteria previously established by the Centers for Disease Control and Prevention (CDC) using XbaI as the restriction enzyme. Briefly, following 18 to 20 h growth on TSA at 37 °C, genomic DNA was digested with 50 U XbaI (TaKaRa, Japan) for 2 h at 37 °C, then the DNA fragments were subsequently separated on a 1.0% SeaKem Gold agarose gel (Lonza, USA) in 0.5× Tris-borate-EDTA (TBE) buffer using a CHEFMapper gel apparatus (Bio-Rad Laboratories, California, USA). The conditions for electrophoresis were as follows: pulse time, 2–30s at 14 °C; run time, 18 h; voltage, 6 V/cm. Analysis of the image was performed by using the Bionumerics software (Applied Maths, Belgium).
5
Genotyping of Integron-Positive A. baumannii
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The integron-positive A. baumannii isolates were genotyped using pulsed-field gel electrophoresis (PFGE) [24 (link)], while Salmonella serotype Braenderup H9812 strain (PulseNet China, Beijing, China) served as a standard quality control isolate. Briefly, genomic DNA was extracted from integron-positive A. baumannii isolates, and digested with restriction endonuclease Apa I (New England Biolabs, Inc., Beverly, MA, USA). Electrophoresis was performed at 14 °C on a 1% Seakem Gold agarose gel (Lonza, Rockland, ME, USA) in the CHEF Mapper® Pulsed Field Electrophoresis System (Bio-Rad, Hercules, CA, USA) for 19 h under the following conditions: switching time of 5 to 20 s, 120° field angle, and voltage of 6 V/cm. Gel images were captured using the Gel Doc XR+ imaging system (Bio-Rad), and all PFGE profiles were processed using the software BioNumerics version 6.0 (Applied Maths, Inc., Austin, TX, USA). Similarities were obtained using the Dice coefficient at a 1.5% tolerance, and a dendrogram was constructed with the unweighted-pair group method using average linkages (UPGMA) clustering method. A mean similarity of <90% indicated various PFGE genotypes, whereas a similarity of 90% or greater was defined as the same PFGE genotype [24 (link)].
6
Plasmid Profiling by S1-PFGE
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Pulsed field gel electrophoresis (S1-PFGE) was performed to determine the number and size of plasmids carried by strain 24835 as described previously. Briefly, agarose plugs containing whole-cell DNA of strain 24835 were treated with 8 U of S1 nuclease (Fermentas, Thermo Scientific, Waltham, MA, USA) and the reaction was stopped by adding 0.5 M EDTA (pH = 8). PFGE was conducted with a 1% SeaKem Gold agarose gel (Lonza, Basal, Switzerland) using a CHEF DRII system (Bio-Rad, Hercules, CA, USA) at 14°C, with a 6-V/cm current and run times of 12 h at switch time of 5–40 s followed by 8 h at switch time of 3–8 s. MidRange I PFG Marker (NEB, Ipswich, MA, USA) was used for size estimation.
7
MRSA Strain Identification via PFGE
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The 18 non-ST398 MRSA isolates were subjected to pulsed-field gel electrophoresis
(PFGE) analysis. Plugs digested with SmaI were run on
1.2% SeaKem Gold agarose gel (Lonza, Basel, Switzerland) with 0.5x
Tris-Borate EDTA (TBE) buffer (Bioneer, Korea) on the CHEF Mapper PFGE system
(Bio-Rad, CA, USA) for 18 h at 14°C, with an initial and final switch
time of 5.16 s and 40.17 s, respectively. Salmonella entericaserovar Braenderup digested with XbaI was used as the molecular
reference marker. Gels were stained with SYBR Gold solution (Invitrogen, CA,
USA), and viewed and recorded under a UV transilluminator. The images were
analyzed using the BioNumerics software (Applied Maths, TX, USA).
(PFGE) analysis. Plugs digested with SmaI were run on
1.2% SeaKem Gold agarose gel (Lonza, Basel, Switzerland) with 0.5x
Tris-Borate EDTA (TBE) buffer (Bioneer, Korea) on the CHEF Mapper PFGE system
(Bio-Rad, CA, USA) for 18 h at 14°C, with an initial and final switch
time of 5.16 s and 40.17 s, respectively. Salmonella entericaserovar Braenderup digested with XbaI was used as the molecular
reference marker. Gels were stained with SYBR Gold solution (Invitrogen, CA,
USA), and viewed and recorded under a UV transilluminator. The images were
analyzed using the BioNumerics software (Applied Maths, TX, USA).
8
Genotypic Diversity of 3GC-Resistant E. coli
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The genotypic diversity of 3GC-resistant E. coli was analysed using multilocus sequence typing (MLST) [34 (link)]. Seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, and recA) were also sequenced. DNA sequence variations were analysed using the MLST database for E. coli [35 ] to determine the sequence types (STs).
The similarity of the isolates was compared using pulsed-field gel electrophoresis (PFGE) analysis according to the manufacturer's protocol (Bio-Rad, Hercules, CA, USA). The whole-cell DNA of E. coli isolates embedded in 1.6% low-melt agarose plugs (Bio-Rad, Hercules, CA, USA) was digested with the XbaI restriction enzyme (Takara Bio Inc., Japan) for 18 h at 37 °C [36 (link)]. DNA fragments were electrophoresed in 1.0% SeaKem Gold agarose gel (Lonza, USA) on the CHEF-MAPPER system (BioRad Laboratories, Japan) with 0.5× Tris-borate-EDTA running buffer at 14 °C for 20 h at 6 V/cm with a pulse time of 5.3–49.9 s, at an angle of 120°. The PFGE patterns were interpreted according to the criteria described by Tenover et al. [37 (link)] as “indistinguishable” isolate if only one band differed; “closely related” isolate if 2–3 bands differed; and “possibly related” isolate if up to six bands differed, from those of the reference strain.
The similarity of the isolates was compared using pulsed-field gel electrophoresis (PFGE) analysis according to the manufacturer's protocol (Bio-Rad, Hercules, CA, USA). The whole-cell DNA of E. coli isolates embedded in 1.6% low-melt agarose plugs (Bio-Rad, Hercules, CA, USA) was digested with the XbaI restriction enzyme (Takara Bio Inc., Japan) for 18 h at 37 °C [36 (link)]. DNA fragments were electrophoresed in 1.0% SeaKem Gold agarose gel (Lonza, USA) on the CHEF-MAPPER system (BioRad Laboratories, Japan) with 0.5× Tris-borate-EDTA running buffer at 14 °C for 20 h at 6 V/cm with a pulse time of 5.3–49.9 s, at an angle of 120°. The PFGE patterns were interpreted according to the criteria described by Tenover et al. [37 (link)] as “indistinguishable” isolate if only one band differed; “closely related” isolate if 2–3 bands differed; and “possibly related” isolate if up to six bands differed, from those of the reference strain.
9
Agarose Gel Electrophoresis of CPP-DNA Complexes
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Various amounts of CPPs were complexed with 2 μg of the pEGFP-N1 plasmid DNA and incubated at 37°C for 1 h. Complexes formed at N/P ratios ranging from 0 (DNA only) to 18 were analyzed by electrophoresis on a 0.5% SeaKem Gold agarose gel (Lonza Group, Basel, Switzerland) in 1 × TBE buffer (89 mM of Tris-borate and 2 mM of EDTA, pH 8.3) at 100 V for 30 min [35 ]. Gels were stained with the SYBR Safe DNA gel stain (Life Technologies), and images were captured using the ChemiDoc XRS+ gel imaging system (Bio-Rad, Hercules, CA, USA) with an excitation wavelength at 302 nm of trans-UV light and with an emission wavelength at 548–630 nm of the standard filter. Data were analyzed using the Quantity One 1-D analysis software 4.6.9 (Bio-Rad). The relevant percentage of a DNA band shift migrating in the gel was defined as a reciprocal mobility ratio between minimal N/P ratio (N/P = 0) setting as 0% and maximal N/P ratio (N/P = 18) setting as 100%.
10
Telomeric DNA Analysis by Pulsed-Field Gel Electrophoresis
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Genomic DNA was extracted using the Qiagen DNeasy Kit (Qiagen) according to the manufacturer’s instructions. A total of 5 μg of DNA was digested overnight with HinfI and RsaI (40 U each). The digested DNA was resolved on a 1% SeaKem Gold agarose gel (Lonza) in 0.5×Tris/Borate/EDTA (TBE) buffer, using a CHEF electrophoresis chamber (BioRad). Electrophoresis was conducted with initial switch time 0.2 s and final switch time 13 s at 6 V/cm for 15 h. The gel was blotted onto a positively charged Biodyne B nylon membrane (Pall, VWR) and hybridized at 42°C in 6 × saline-sodium citrate buffer (SSC), 0.01% sodium dodecyl sulphate (SDS) with 20 pmol of digoxigenin (DIG)-labeled telomeric C-rich oligonucleotide TAA(CCCTAA)4 prepared using the 3′ end labeling kit (Roche, 03353575910). Following hybridization washes (twice 5 min in 2 × SSC, 0.01% SDS and once 2 min in 0.1 × SSC, 0.01% SDS) the signal was revealed using the anti-DIG-alkaline phosphatase antibodies (Roche, 11093274910) and CDP-Star (Roche) following the manufacturer’s instructions. Images were obtained using the Luminescent image analyzer LAS-4000 mini (GE Healthcare) and analyzed using the Telometric 1.2 software.
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