U-2-OS cells were transfected with siRNA for 24 hours and treated with UCN-01 for 4 hours or irradiated with 20 Gy of ionizing radiation. Cells (1 × 106) were molded into 1% low-melting agarose plugs (InCert Agarose, Lonza), followed by treatment with proteinase K buffer [10 mM tris-Cl (pH 7.5), 50 mM EDTA, 1% N-laurylsarcosyl, proteinase K (2 mg/ml)] at 50°C for 48 hours. Plugs were then subjected to PFGE (1% agarose, CHEF-DR II system; Bio-Rad Laboratories; 120° angle, 60- to 240-s switch time, and 4 V/cm) for 20 hours. DNA was visualized by ethidium bromide staining. Quantification was performed using ImageJ software.
Incert agarose
Manufactured by Lonza
Sourced in United States, Switzerland
InCert agarose is a high-quality laboratory-grade agarose product manufactured by Lonza. It is designed for use in various gel electrophoresis applications, providing reliable and consistent results. The core function of InCert agarose is to serve as a separation medium for the analysis and purification of biomolecules, such as DNA, RNA, and proteins.
Automatically generated - may contain errors
Lab products found in correlation
Chef dr 2 system, by Bio-Rad (5 mentions)
Seakem gold agarose, by Lonza (4 mentions)
Chef dr 3, by Bio-Rad (4 mentions)
Driselase, by Merck Group (3 mentions)
Glucanex, by Merck Group (3 mentions)
Potato dextrose broth (pdb), by BD (3 mentions)
G box gel imaging system, by Syngene (3 mentions)
Chromosomal grade agarose, by Bio-Rad (2 mentions)
Xbai, by Takara Bio (1 mentions)
S1 nuclease, by Takara Bio (1 mentions)
Dig high prime dna labeling and detection starter kit 2, by Roche (1 mentions)
Tbe buffer, by Merck Group (1 mentions)
Smai enzyme, by Thermo Fisher Scientific (1 mentions)
Ethidium bromide, by Merck Group (1 mentions)
Fluorchemq multiimage 3, by Bio-Techne (1 mentions)
Proteinase k, by Merck Group (1 mentions)
14 protocols using incert agarose
1
DNA Damage Assessment via PFGE
2
Plasmid Profiling of Vancomycin-Resistant Enterococci
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
S1-PFGE was performed as described previously [2 ]. Overnight incubated cells of Efm4, Efs2 and transconjugants were embedded in InCert Agarose (Lonza, USA), respectively, followed by digestion with S1 nuclease (TaKaRa, Japan). The DNA restriction fragment were separated in 1% SeaKem Gold® Agarose (Lonza, USA) using a pulse gel electrophoresis apparatus (Bio-Rad, USA). Genomic DNA of Salmonella serovar Braenderup strain H9812, digested with XbaI (TaKaRa, Japan), was used as a molecular standard.
Southern hybridization was performed on the S1-PFGE gel using DIG High Prime DNA Labeling and Detection Starter Kit II (Roche, Germany) with digoxin- labeled DNA probes specific for vanA, followed by manufacturer’s protocol.
Southern hybridization was performed on the S1-PFGE gel using DIG High Prime DNA Labeling and Detection Starter Kit II (Roche, Germany) with digoxin- labeled DNA probes specific for vanA, followed by manufacturer’s protocol.
3
Fusarium Protoplast CHEF Electrophoresis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protoplasts from Fusarium strains were prepared as described previously (Tudzynski et al. 1999 (link)). The protoplast suspension was mixed with 1.2% InCert agarose (Lonza Group AG, Basel, Switzerland) and then loaded on a CHEF (contour-clamped homogeneous electric field) gel as described in Crawford et al. (2008) (link). Chromosomes of Schizosaccharomyces pombe and Saccharomyces cerevisiae were used as a molecular size marker (Bio-Rad, Munich, Germany).
4
Pulsed-Field Gel Electrophoresis of SmaI-Digested Genomic DNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Genomic DNA was prepared in situ in agarose blocks (InCert Agarose, Lonza, Walkersville, MD, USA) as described previously (Hung and Bandziulis, 1990) and was digested overnight with the restriction enzyme SmaI (Fermentas, Vilnius, Lithuania) at 30°C (Howard et al., 1992) . The SmaI-generated DNA fragments were resolved by PFGE in 1% agarose (PFGE-certified, Bio-Rad, Hercules, CA, USA) and 0.5x TBE buffer (Merck, Darmstadt, Germany) using a CHEF DR III (Bio-Rad, Hercules, CA, USA) apparatus.
Electrophoresis was performed at a constant voltage of 6 V/cm and a temperature of 14°C for 23 h with a 120° angle and a pulse time of 1 to 25 s. The Lambda DNA ladder (BioLabs, Hitchin, UK) was used as a molecular size marker. The PFGE DNA patterns were compared, and a dendrogram was constructed using SynGene GeneTools, File version 4.00.00 (SynGene, Cambridge, UK).
Electrophoresis was performed at a constant voltage of 6 V/cm and a temperature of 14°C for 23 h with a 120° angle and a pulse time of 1 to 25 s. The Lambda DNA ladder (BioLabs, Hitchin, UK) was used as a molecular size marker. The PFGE DNA patterns were compared, and a dendrogram was constructed using SynGene GeneTools, File version 4.00.00 (SynGene, Cambridge, UK).
5
Quantifying Double-Strand DNA Breaks
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To identify double strand breaks (DSBs) in DNA 1 × 106 cells were treated with either DMSO or 0.5 μM complex 1 with and without 405 nm light exposure, as described above. Cells were resuspended in 50 μl PBS and mixed with 50 μl InCert agarose (Lonza, Basel, Switzland). The mixture was then added to the plug insert and allowed to set at 4 °C for 20 minutes. Plugs were then incubated in 1 ml of 0.5 M EDTA, 1% N-laurylsarcosyl, proteinase K (1 mg/ml) buffer for 48 h at room temperature. Following incubation, plugs were washed 4 times in TE buffer and then loaded into a 0.7% chromosomal grade agarose gel. Separation by pulse field electrophoresis was subsequently performed for 24 h (Biorad; 120o angle, 60–240 s switch time, 4 V/cm). After pulse field electrophoresis the gel was stained with EtBr and visualised on a UGenius fluorescence gel imager (Syngene, Cambridge, UK).
6
Fungal Chromosome Separation by CHEF Electrophoresis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To confirm HCT, CHEF electrophoresis was performed. Preparation of protoplasts and pulsed‐field gel electrophoresis were performed as described previously (Ma et al., 2010 ). Fusarium strains were cultured in 100 ml NO3 medium (0.17% yeast nitrogen base, 100 mM KNO3 and 3% sucrose) for 5 days at 25°C. Then, conidia were collected by filtering through a double‐layer of miracloth and the concentration of spores were measured. 5 × 108 conidia were transferred to 40 ml PDB (BD biosciences). After approximately 16 h of growth at 25°C, germinated spores were re‐suspended in 10 ml MgSO4 solution (1.2 M MgSO4, 50 mM sodium citrate, pH 5.8) supplemented with 50 mg ml−1 Glucanex (Sigma) + 5 mg ml−1 driselase (Sigma, D9515) and incubated for approximately 17 h at 30°C in a shaking water bath (65 r.p.m.). The protoplasts were filtered through a double layer of miracloth, collected by centrifugation and casted in InCert agarose (Lonza) at a concentration of 2 × 108 protoplasts ml−1. Plugs were treated with 2 mg ml−1 pronase E at 50°C. Chromosomes were separated by pulsed‐field electrophoresis for 260 h in 1% Seakem Gold agarose (Lonza) at 1.5 V cm−1 in a CHEF‐DRII system (Bio‐Rad) in 0.5 × TBE at 4°C, with switch times between 1200 and 4800 s. The gels were stained with 1 μg ml−1 ethidium bromide in 0.5 × TBE.
7
Protoplast Preparation and Pulsed-Field Gel Electrophoresis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Preparation of protoplasts and pulsed‐field gel electrophoresis were performed as described previously (van Dam et al., 2017 ). Fusarium strains were cultured in 100 ml NO3 medium (0.17% yeast nitrogen base, 100 mM KNO3, and 3% sucrose) for 5 days at 25 °C. Then, conidia were collected by filtering through a double layer of Miracloth and the concentration of spores was measured. Conidia (5 × 108) were transferred to 40 ml PDB (BD Biosciences). After approximately 16 hr of growth at 25 °C, germinated spores were resuspended in 10 ml of MgSO4 solution (1.2 M MgSO4, 50 mM sodium citrate, pH 5.8) supplemented with 50 mg/ml Glucanex (Sigma) + 5 mg/ml driselase (Sigma) and incubated for approximately 17 hr at 30 °C in a shaking water bath (65 rpm). The protoplasts were filtered through a double layer of Miracloth, collected by centrifugation and, cast in InCert agarose (Lonza) at a concentration of 2 × 108 protoplasts per millilitre. Plugs were treated with 2 mg/ml pronase E at 50 °C. Chromosomes were separated by pulsed‐field electrophoresis for 260 hr in 1% Seakem Gold agarose (Lonza) at 1.5 V/cm in a CHEF‐DRII system (Biorad) in 0.5 × Tris‐borate‐EDTA (TBE) buffer at 4 °C, with switch times between 1,200 and 4,800 s. The gels were stained with 1 μg/ml ethidium bromide in 0.5 × TBE.
8
Chromosome Transfer Confirmation via CHEF
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To confirm horizontal chromosome transfer, Contour-clamped homogeneous electric field (CHEF) electrophoresis was performed. Preparation of protoplasts and pulsed-field gel electrophoresis were performed as described previously (4). Fusarium strains were cultured in 100 ml NO 3 medium (0.17% yeast nitrogen base, 100 mM KNO 3 and 3% sucrose) for five days at 25 °C. Then, conidia were collected by filtering through a double-layer of miracloth and the concentration of spores were measured. Five 10 8 conidia were transferred to 40 ml PDB (BD biosciences). After approximately 16 hours of growth at 25 °C, germinated spores were resuspended in 10 ml MgSO 4 solution (1.2 M MgSO 4 , 50 mM sodium citrate, pH 5.8) supplemented with 50 mg/ml Glucanex (Sigma) + 5 mg/ml driselase (Sigma, D9515) and incubated for approximately 17 hours at 30°C in a shaking water bath (65 rpm). The protoplasts were filtered through a double layer of miracloth, collected by centrifugation and casted in InCert agarose (Lonza) at a concentration of 2 10 8 protoplasts per ml. Plugs were treated with 2 mg/ml pronase E at 50°C. Chromosomes were separated by pulsed-field electrophoresis for 260 hours in 1% Seakem Gold agarose (Lonza) at 1.5 V/cm in a CHEF-DRII system (Biorad) in 0.5 × TBE at 4 °C, with switch times between 1200 and 4800 s. The gels were stained with 1g/mL ethidium bromide in 0.5 × TBE.
9
Pulsed-Field Gel Electrophoresis of E. cecorum
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PFGE was done as previously described [26 (link), 34 (link)–36 (link)]. Genomic DNA from all isolates was embedded in 2% agarose plugs (InCert Agarose, Lonza, Rockland, USA). DNA was digested with the SmaI enzyme (20 U/μl; Fermentas, Lithuania). Electrophoresis of digested fragments was carried in 1% agarose on CHEF DRII system (Bio-Rad Laboratories, Berkeley, CA, USA) using 0.5xTBE at 14°C. The initial and final switch time was 0.5 and 35 s respectively, at 6V/min for 24 h. The DNA banding patterns were analysed with Gel Compar II BioNumerics v. 7.0 software (Applied Maths, Belgium) and cluster analysis was performed by UPGMA based on the Dice similarity coefficient, with optimization and position tolerance set at 1%. EC isolates were clustered using > 80% homology cut-off, above which strains were considered to be closely related and assigned to the same PFGE type. The reference strain E. cecorum ATCC 43198 was used as control.
10
Detecting DNA Double-Strand Breaks
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To detect DSBs, 2 × 106 cells per sample were treated as indicated, harvested, and melted into 1.0% InCert-Agarose (Lonza) inserts. Inserts were digested in 0.5 M EDTA-1% N-laurylsarcosyl-proteinase K (1 mg/ml) at room temperature for 48 h and washed three times in TE buffer. Inserts were loaded onto a separation gel (1.0% chromosomal-grade agarose, Bio-Rad). The separation was performed using a CHEF DR III (BioRad; 120 field angle, 240 s switch time, 4 V cm−1, 14 °C) for 20 h. Images of ethidium bromide-stained gels were acquired using a Syngene G:BOX gel imaging system. DSBs (chromosome fragments >2 Mbp) were quantified by densitometry using ImageJ and normalized to the total amount of DNA in the gel.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!