For strain typing, whole-cell E. coli DNA embedded in agarose gel plugs was digested with XbaI (Takara Bio Inc.) and was separated by Pulse-field gel electrophoresis (PFGE) using a CHEF-DR III apparatus (Bio-Rad, Hercules, CA, United States), as previously described (Beutin et al., 2005 (link)). Plasmids were analyzed and sized using the S1-PFGE nuclease method. Large fragments from the restriction enzyme digest were separated by PFGE using a CHEF-DR III apparatus (Bio-Rad) for 7 h at 6 V/cm and 14°C, with initial and final pulse times of 5 and 20 s, respectively. Southern hybridization was performed with the blaKPC–2 gene as a probe to ascertain the blaKPC–2-positive plasmid, using the electrochemiluminescence direct nucleic acid labeling and detection system per the manufacture’s operation protocol (GE Healthcare, Buckinghamshire, United Kingdom).
Chef dr 3 apparatus
Manufactured by Bio-Rad
Sourced in United States, France
The CHEF-DR III apparatus is a pulsed-field gel electrophoresis (PFGE) system designed for the separation and analysis of large DNA molecules. The CHEF-DR III allows for the resolution of DNA fragments ranging from 50 kilobase pairs (kbp) to 6 megabase pairs (Mbp).
Automatically generated - may contain errors
Lab products found in correlation
Xbai, by Takara Bio (4 mentions)
Pulse field certified agarose, by Bio-Rad (3 mentions)
Fpquest software, by Bio-Rad (3 mentions)
Fpquest software v4, by Bio-Rad (2 mentions)
Xbai enzyme, by New England Biolabs (2 mentions)
Pulsed field certified agarose, by Bio-Rad (2 mentions)
Agarose gel, by Bio-Rad (2 mentions)
45u xbal, by Takara Bio (2 mentions)
Dryslide, by BD (1 mentions)
Gel doc xr software, by Bio-Rad (1 mentions)
Disposable plug mold, by Bio-Rad (1 mentions)
70 μm cell strainer, by BD (1 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (1 mentions)
Alpha imager, by Bio-Techne (1 mentions)
Spei restriction enzyme, by Thermo Fisher Scientific (1 mentions)
Chef bacterial genomic dna plug kit, by Bio-Rad (1 mentions)
Smai enzyme, by Thermo Fisher Scientific (1 mentions)
Pfge certified agarose, by Bio-Rad (1 mentions)
Fingerprinting 2 informatix software, by Bio-Rad (1 mentions)
Lysozyme solution, by Thermo Fisher Scientific (1 mentions)
64 protocols using chef dr 3 apparatus
1
Strain Typing and Plasmid Analysis of E. coli
2
Detecting and Typing Vancomycin-Resistant Enterococci
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Bacti-swabs (Remel, Lenexa, KS) were used by trained research personnel to obtain patient and environmental samples. Patients’ nares, oral cavity, groin, and perianal site swabs were cultured on bile-esculin plates with 6 mg/L vancomycin (BEV6) on the same day. Hands and environmental swabs (bed controls, side table top and bottom, nurse call button, curtain, toilet seat, door knob, TV remote control, wheelchair, and other equipment when available) were enriched overnight in Brain Heart Infusion broth at 36ºC before culturing on BEV6 plates. Growth suggestive of VRE was confirmed by pyrrolidonyl arylamidase testing (DrySlide; BD, Franklin Lakes, NJ).
Pulsed-field gel electrophoresis was performed to determine the relatedness of VRE isolates. Genomic deoxyribonucleic acid was prepared and digested with SmaI (New England BioLabs, Beverly, MA) using a previously described method [14 (link)]. SmaI fragments were separated using a CHEF DR III apparatus (Bio-Rad, Hercules, CA) and compared using BioNumerics software (Applied Maths, Kortrijk, Belgium). Similarity of isolates was calculated using Dice coefficient (BioNumerics software). Isolates were placed in the same pulsotype if their restriction patterns were ≥80% similar [14 (link)].
Pulsed-field gel electrophoresis was performed to determine the relatedness of VRE isolates. Genomic deoxyribonucleic acid was prepared and digested with SmaI (New England BioLabs, Beverly, MA) using a previously described method [14 (link)]. SmaI fragments were separated using a CHEF DR III apparatus (Bio-Rad, Hercules, CA) and compared using BioNumerics software (Applied Maths, Kortrijk, Belgium). Similarity of isolates was calculated using Dice coefficient (BioNumerics software). Isolates were placed in the same pulsotype if their restriction patterns were ≥80% similar [14 (link)].
3
Double-Strand Break Detection by PFGE
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DSB detection by PFGE was done as reported previously18 (link). Briefly, cells were cast into 0.8% agarose plug (2.5 x 105 cells/plug), digested in lysis buffer (100 mM EDTA, 1% sodium lauryl sarcosine, 0.2% sodium deoxycholate, 1 mg/ml proteinase-K) at 37 °C for 36–40 h, and washed in 10 mM Tris-HCl (pH 8.0)–100 mM EDTA. Electrophoresis was performed at 14 °C in 0.9% pulse field-certified agarose (Bio-Rad) using Tris-borate-EDTA buffer in a Bio-Rad Chef DR III apparatus (9 h, 120°, 5.5 V/cm, and 30- to 18-s switch time; 6 h, 117°, 4.5 V/cm, and 18- to 9-s switch time; and 6 h, 112°, 4 V/cm, and 9- to 5-s switch time). The gel was stained with ethidium bromide and imaged on Uvidoc-HD2 Imager. Quantification of DSB was carried out using ImageJ software64. Relative DSB levels were calculated by comparing the results in the treatment conditions to that of the DSB level observed in untreated controls.
4
Pulsed-Field Gel Electrophoresis of Bacterial DNA
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Pulsed-field gel electrophoresis (PFGE) was performed as previously described [41 (link)]. In brief, The DNAs of PM1 and PM1-1 to PM1-5 were digested with SmaI (New England BioLabs, Ipswich, MA) and then were separated using a CHEF-DRIII apparatus (Bio-Rad Laboratories). PFGE was carried out at 200 V and 12°C for 20 h with the pulse times ranging from 5 to 60 s.
5
Pulsed-field Gel Electrophoresis of Non-O157 E. coli
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PFGE was performed according to the PulseNet protocol developed for non-O157 E. coli (http://www.cdc.gov/pulsenet/PDF/ecoli-shigella-salmonella-pfge-protocol-508c.pdf ). The genomic DNA was digested with 45 U of XbaI (Takara, China) at 37°C for 2 h. Macrorestriction fragments were resolved by counter-clamped homogeneous electric field electrophoresis in a CHEF-DRIII apparatus (Bio-Rad, USA). The run time was 19 h at 6.0 V/cm, with initial and final switch times of 6.76 s and 35.38 s, respectively. The image was captured with a Gel Doc XR+ software (Bio-Rad, USA). Data analysis was performed and an UPGMA dendrogram was constructed using Bionumerics (Version 4.0, Applied Maths BVBA, Belgium).
6
Pulsed-field Gel Electrophoresis for DNA Damage
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Sub-confluent cultures of U2OS were treated with vehicle alone (DMSO), camptothecin (CPT 1 μM), or Ru65 (50 μM) and were either non-irradiated or UV-A irradiated. Cells were harvested by trypsinization, and agarose plugs of 106 cells were prepared in a disposable plug mold (Bio-Rad). Plugs were incubated in lysis buffer (100 mM EDTA, 1% (w/v) sodium lauryl sarcosyl, 0.2% (w/v) sodium deoxycholate, 1 mg ml–1 proteinase K) at 37 °C for 72 h, and washed four times in 20 mM Tris–HCl pH 8.0, 50 mM EDTA before loading onto an agarose gel. Electrophoresis was performed for 23 h at 14 °C in 0.9% (w/v) Pulse Field Certified Agarose (Bio-Rad) containing Tris-borate/EDTA buffer according to the conditions described in47 (link) and adapted to the Bio-Rad CHEF DR III apparatus. The gel was finally stained with ethidium bromide (EtBr) and analyzed using an Alpha Innotech Imaging system.
7
Pulsed-field Gel Electrophoresis for VRE Profiling
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Genomic DNA was isolated, digested with restriction endonuclease SmaI, and treated as previously described13 (link). The agarose gel concentration was 1% and the CHEF-DR III apparatus (BIO-RAD laboratories, Hercules, CA, USA) was used for pulsed field gel electrophoresis (PFGE). Selected ramped pulsed times were as follows: 1–11 sec for 15 h and 11–30 sec for 14 h at 14 °C. Relatedness between banding patterns was calculated using a band based similarity coefficient (Dice) and UPMGA clustering (BioNumerics, Applied Maths, Sint-Martens-Latem, Belgium). A composite tree of all 58 hospital VRE of groups I–III resolved in 18 independent PFGE gels was generated the same way with the exception of increasing the value of the “position tolerance setting” to 1.5% (default: position tolerance setting 1.0%; optimization 0.5%). PFGE types were assigned based on >90% similar patterns and additionally considering recommendations for fragment pattern analyses as previously described14 (link)15 (link).
8
Genetic Relatedness Analysis by PFGE
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Genetic relatedness of the isolates was examined by PFGE in a CHEF-DRIII apparatus (Bio-Rad Laboratories, Hercules, and CA) following digestion of genomic DNA with XbaI enzyme (New England Biolabs, Massachusetts) according to Tenover et al. [20 (link)]. The PFGE images were processed and the dendrogram was calculated by FPQuest software v4.5 (Biorad laboratories inc, Hercules, California, USA.) using Dice coefficient and UPGMA (unweighted pair group method using arithmetic averages). Isolates having more than 95% similarity were considered identical.
9
Characterizing E. coli and K. pneumoniae Isolates
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E. coli and K. pneumoniae isolates were characterized by multi-locus sequence typing (MLST), as previously described (Diancourt et al., 2005 (link); Wirth et al., 2006 (link)). E. coli isolates belonging to the same sequence type (ST) and recovered from child and parent isolates from the same household, were assessed for genetic relatedness by PFGE of XbaI-digested genomic DNA using a CHEF DR-III apparatus (Bio-Rad Laboratories, Hercules, CA, USA) following the standardized protocol of PulseNet (Ribot et al., 2006 (link)). XbaI-digested genomic DNA from Salmonella enterica serotype Braenderup strain H9812 was used as a molecular reference marker (Hunter et al., 2005 (link)). Cluster analysis was performed using BioNumerics, version 6.6 (Applied Maths, Sint-Martens-Latem, Belgium) as previously described (Liakopoulos et al., 2016 (link)).
10
Pulsed-field Gel Electrophoresis of Liver Tissue
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PFGE was performed as published previously (Neelsen et al., 2013 (link)). Briefly, snap-frozen liver tissue was directly put into 4% formaldehyde without thawing and incubated for 10 min at 37°C. Tissue was mechanically dissociated (gentleMACS Dissociator, Miltenyi Biotec), filtered through a 70 μm cell strainer (Falcon) and 2.5x105 cell were embedded in a 0.8% agarose plus, digested in lysis buffer (100 mM EDTA, 1% (wt/vol) sodium lauryl sarcosyne, 0.2% (wt/vol) sodium deoxycholate, and 1 mg/ml proteinase K) at 37°C for 48 h, and washed in 10 mM Tris-HCl, pH 8.0, and 100 mM EDTA. Electrophoresis was performed at 14°C in 0.9% (wt/vol) Pulsed Field Certified Agarose (Bio-Rad Laboratories) containing Tris-borate/EDTA buffer in a CHEF DR III apparatus (9 h, 120°, 5.5 V/cm, 30-18 s switch time; 6 h, 117°, 4.5 V/cm, 18-9 s switch time; 6 h, 112°, 4 V/cm, 9-5 s switch time; Bio-Rad Laboratories). The gel was stained with ethidium bromide and imaged on an Alpha Innotech Imager.
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