Snap-frozen L3s were thawed and used to make agarose plugs using the CHEF Genomic DNA Plug Kit (Bio-Rad). Approximately 3 × 103 L3s were spun at 1,000g for 5 min, resuspended in 32 μl cell suspension buffer and incubated in a 50 °C water bath; 53 μl of 2% clean-cut agarose, melted at 50 °C was added to the L3 and mixed gently before being transferred to a plug mould and stored at 4 °C. The plug was incubated in 100 μl proteinase K and 2.5 μl proteinase K reaction buffer at 50 °C for 1 day, before being washed five times, 1 h per wash, with gentle agitation, in 1× Wash buffer. The plug was stored at 4 °C until use. For optical mapping, DNA molecules were stretched and immobilized along microfluidic channels before digestion with the restriction endonuclease SpeI, yielding a set of restriction fragments ordered by their location in the genome. The fragments were fluorescently stained and visualized to determine the fragment sizes. Assembling overlapping fragment patterns of single-molecule restriction maps produced an optical map of the genome consisting of six large optical contigs with a total size of 92.54 Mb.
Chef genomic dna plug kit
Manufactured by Bio-Rad
Sourced in United States
The CHEF Genomic DNA Plug Kit is a laboratory product designed for the preparation of high-molecular-weight genomic DNA samples. The kit provides a simple and efficient method for isolating intact, high-quality genomic DNA from a variety of cell types. The DNA samples prepared using this kit are suitable for downstream applications such as pulsed-field gel electrophoresis (PFGE).
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Lab products found in correlation
Gelase, by Illumina (3 mentions)
Chef dr 3 system, by Bio-Rad (3 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (3 mentions)
Lysis buffer, by Bionano Genomics (2 mentions)
Chef mapper xa pulsed field electrophoresis system, by Bio-Rad (2 mentions)
Chef dr 2 system, by Bio-Rad (2 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
Chef drii apparatus, by Bio-Rad (2 mentions)
Certified megabase agarose, by Bio-Rad (2 mentions)
Bionano prep blood dna isolation protocol, by Bionano Genomics (1 mentions)
Agarase, by Thermo Fisher Scientific (1 mentions)
Dialysis membrane, by Merck Group (1 mentions)
Proteinase k, by Qiagen (1 mentions)
Rbc lysis solution, by Qiagen (1 mentions)
Puregene proteinase k, by Qiagen (1 mentions)
Millipore membrane filter, by Merck Group (1 mentions)
Qubit dsdna assay kit, by Thermo Fisher Scientific (1 mentions)
Quant it dsdna assay kit, by Thermo Fisher Scientific (1 mentions)
Sigmagel software, by Merck Group (1 mentions)
Ultrapure agarose, by Thermo Fisher Scientific (1 mentions)
26 protocols using chef genomic dna plug kit
1
Optical Mapping of Nematode Genome
2
High-Molecular-Weight DNA Extraction
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The C666-1 cell line was washed with phosphate-buffered saline (PBS) and spun down to a pellet. Next, 106 cells/mL were obtained upon resuspension in PBS, and embedded in 1.5 % low-melting agarose plugs in 0.5 × TBE (Tris-Borate-EDTA) (CHEF Genomic DNA Plug Kit, Bio-Rad). Subsequent handling of the DNA followed BioNano Genomics recommended protocols. The agarose plugs were incubated with proteinase K with lysis buffer at 50 °C overnight. The plugs were washed with a wash buffer to stabilize the DNA in the plugs, and the quality was assessed using pulsed-field gel electrophoresis. A plug was then washed with TE (Tris-EDTA) buffer and melted at 70 °C. After being solubilized with 0.4 U of GELase (Epicentre), the purified DNA was subjected to 2.5 h of drop-dialysis and was shredded by nine strokes of gentle pipetting. The viscous DNA was allowed to equilibrate overnight at room temperature to increase the homogeneity. It was then quantified using a Qubit Broad Range dsDNA Assay Kit (Life Technologies).
3
Separation of Intact Chromosomal DNA
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In order to determine the size of intact chromosomes of DDNA#1, a BioRad CHEF Genomic DNA Plug Kit was used. Briefly, yeast cells were treated with lyticase and the resulting spheroplasts were embedded in low melting point agarose. After incubation with RNase A and Proteinase K, the agarose plugs were thoroughly washed in TE. The DNA in the agarose plugs was separated on a 0.88% agarose gel in 1xTAE buffer on a Bio-Rad CHEF DRII system. The DNA was separated in four subsequent 12 hour runs at 3V/cm; run one and two used a constant switching time of 500 seconds, and in run three and four the switching time increased from 60 seconds to 120 seconds. The gel was afterwards stained with ethidium bromide and imaged.
4
Isolation and Extraction of High-Quality DNA from Diverse Biological Samples
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Peripheral blood and culture cells DNA were isolated and extracted according to the Bionano Prep Blood DNA Isolation Protocol (Bionano Genomics #30033). Clinical centrifugation for 3 min at 2000 g was used to separate the white blood cells (WBC) from 3 ml of fresh blood, and these were then resuspended in Cell Suspension Buffer (CSB). High molecular weight DNA of aborted fetal tissue was extracted using the Bionano Prep Animal Tissue DNA Isolation Fibrous Tissue Protocol, (Bionano Genomics #30071). 50 mg tissue was ground up with liquid nitrogen in 2.5 ml of Animal Tissue Homogenize Buffer (HB) and 2.5 ml of anhydrous ethanol (Bionano Genomics). The mixture was left on ice for 1 h, centrifuged, and resuspended in 2 mL HB. The suspension was embedded using a 2% CHEF Genomic DNA Plug Kit (Bio‐Rad) to avoid fracture of DNA molecules. The DNA plugs were incubated in 2.5 ml Lysis Buffer (Bionano Genomics, USA) containing 167 μl of proteinase K (Qiagen) at 50oC overnight, then washed with wash buffer and TE buffer (Bionano Genomics, USA). 2 μl of 0.5 U/μl Agarase (Thermo Fisher) enzyme was added to the plugs into a 1.5 ml microcentrifuge tube and the samples left to stand for 45 min at 43oC. The DNA mixture was dropped into the center of 0.1 μm Dialysis Membrane (EMD Millipore, USA) for 45 min to dry at room temperature.
5
Molecular Epidemiological Analysis of Bacterial Isolates
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All isolates were subjected to molecular epidemiological analysis by PFGE after SmaI digestion using the CHEF Genomic DNA Plug kit (Bio-Rad) according to a standardized protocol [21 (link)]. PFGE was run using a CHEF DRIII system (Bio-Rad). The InfoQuest FP (v5) software (Bio-Rad Laboratories) was used to analyze PFGE profiles, according to interpretation criteria described by Tenover et al. [22 (link)]. Clustering analysis was achieved using Dice similarity coefficients and the unweighted pair group method with averages (UPGMA) at 1.5% optimization and 1.5% position tolerance. Additionally, isolates were typed based on sequencing of the hypervariable region of the S. aureus protein A gene (spa); spa types were assigned MLST sequence types (ST) inferred on the Ridom spaServer (http://spaserver.ridom.de ) curated by the SeqNet.org initiative [23 (link)].
6
High-Molecular-Weight DNA Extraction from Blood
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High molecular weight DNA was extracted both from fresh (<5 days old) and frozen (– 80 °C) whole blood. DNA extraction was performed following the manufacturer’s guidelines (PlugLysis, Bionano Genomics, USA). RBC lysis solution (Qiagen) was used to lyse red blood cells and pellet white blood cells. The white blood cells were re-suspended in cell suspension buffer (Bio-Rad) and embedded into agarose plugs (CHEF Genomic DNA Plug Kit, Bio-Rad) to lessen fragmentation of long DNA molecules during the overnight lysis at 50 °C using a 16:1 ratio of lysis buffer (Bionano Genomics, USA) and Puregene Proteinase K (Qiagen). The plugs were washed with Tris-EDTA buffer and digested at 43 °C with GELase (Epicentre). Extracted high molecular weight DNA was purified from digested materials/enzymes via drop dialysis using Millipore membrane filters (EMD Millipore, USA) placed on Tris-EDTA buffer. DNA quantifications were carried out using Qubit dsDNA assay kits with a Qubit 3.0 Fluorometer (ThermoFisher Scientific).
7
High-Quality Genomic DNA Extraction
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Cells from the trio cell line were washed with PBS, resuspended in cell suspension buffer, and embedded in thin low-melting-point agarose layers (CHEF Genomic DNA Plug Kit, Bio-Rad). The thin agarose layers were incubated with lysis buffer and proteinase K for 4 hr at 50°. The plugs were washed and then solubilized with GELase (Epicentre). The purified DNA was subjected to 4 hr of drop-dialysis. It was quantified using Nanodrop 1000 (Thermal Fisher Scientific) and/or a Quant-iTdsDNA Assay Kit (Invitrogen/Molecular Probes), and the quality was assessed using pulsed-field gel electrophoresis.
8
Yeast Chromosome Separation by PFGE
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Chromosomal DNA was isolated from the remaining 5 mL of yeast cells solutions, prepared as described in protein preparation. DNA isolation was conducted using a CHEF Genomic DNA Plug kit (Bio-Rad, Hercules, CA, USA) according to the methods described by Schwartz and Cantor [37 (link)]. Chromosomes were separated by pulsed field gel electrophoresis (PFGE) in 0.8% agarose gel by means of a CHEF-DR II apparatus (Bio-Rad). Electrophoresis was performed in 0.5× TBE buffer (45 mM Tris, 45 mM boric acid, and 10 mM EDTA; pH 8.2) at 12 °C, and the pulses were as follows: 120 s for 24 h and 240 to 360 s for 24 h, all at 4.5 V/cm. Separated chromosomes were stained in ethidium bromide (0.5 µg/mL) for 15 min with gentle agitation. The molecular weight of bands was estimated using SigmaGel software (Sigma-Aldrich, Gillingham, UK).
9
Telomere Length Analysis in Mouse Embryonic Fibroblasts
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MEFs (5 × 106) were embedded in agarose using a CHEF Genomic DNA Plug kit (Bio-Rad) and digested overnight with excess amounts of restriction enzymes MboI. Cells in agarose were loaded on 1% agarose gels and separated by electrophoresis in 0.5 × TBE, Resolved DNA was transferred to Biodyne B membrane (Pall Biosupport) by capillary transfer. The membrane was hybridized overnight in hybridization buffer (7% sodium dodecyl sulfate, 1% BSA, 0.5 M phosphate [pH 7.2], and 1 mM EDTA) at 42°C with 32P-labeled oligonucleotide telomeric probes (Supplementary Table S1 ). Signals were visualized and quantitated on an Image Analyzer 2500 (Fuji).
10
Chromosomal DNA Isolation and Separation for R. miehei
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Chromosomal DNA of R. miehei CAU432 was prepared as described by Orbach et al. [54 (link)]. Sporangiospores of R. miehei CAU432 obtained from young slant cultures were germinated in a complete medium (containing 0.1% yeast extract, 0.5% tryptone, 1% glucose) at 43°C, followed with isolation of germline protoplasts for chromosome analysis. Chromosomal DNA was prepared in agarose plugs with the CHEF Genomic DNA Plug kit (BioRad) following the instructions of the manufacturer. A 0.6% agarose gel in 0.5 × modified TBE (0.1 M Tris, 0.1 M Boric acid, 0.2 mM EDTA) was used to separate the chromosomes. Chromosome gel electrophoresis of contour-clamped homogeneous electric field (CHEF) was performed using the CHEF Mapper® XA Pulsed Field Electrophoresis System (Bio-Rad) in 0.6% UltraPure™ agarose (Invitrogen) gels at 16°C in circulating 0.5 × TBE buffer, 1.5 V/cm with different pulse intervals for 96 h. After separation, gels were stained in 0.5 μg/ml ethidium bromide for 1 h and then photographed under UV illumination.
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