Chromosomal grade agarose

Manufactured by Bio-Rad

Chromosomal grade agarose is a high-purity agarose product designed for electrophoresis applications, particularly the separation and analysis of large DNA molecules such as chromosomes or megabase-sized DNA fragments. It provides a stable and consistent matrix for the resolution of high-molecular-weight DNA samples.

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Lab products found in correlation

G box gel imaging system, by Syngene (2 mentions) Chef dr 3, by Bio-Rad (2 mentions) Incert agarose, by Lonza (2 mentions) Chef drii apparatus, by Bio-Rad (2 mentions) Chef genomic dna plug kit, by Bio-Rad (1 mentions) Zymolyase 20t, by Seikagaku (1 mentions)

Close spelling variants of this product

Chromosomal grade Agarose

4 protocols using chromosomal grade agarose

Pulsed-Field Gel Electrophoresis for Cellular DNA

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Electrophoresis was performed using a CHEF DR II-apparatus (Biorad, Lunteren, the
Netherlands) with a hexagonal array of 24 electrodes producing a field
reorientation angle of 120° ⁴. Plugs made from 2% (v/v %) agarose (chromosomal grade agarose; Biorad)
containing cells (~ 1.5–2 × 10⁷ cells/ml), lysed [0.5 M EDTA, 1%
Sarcosyl (pH 9), 0.5 mg ml⁻¹ proteinase K for 2 days at 50°C] and
inserted into 0.8% gel agarose (chromosomal grade agarose; Biorad) in
Tris-borate-EDTA (TBE; pH 8). Gels ran at 120° in TBE at 14°C for 24 h using a
linear pulse time of 75 min and a field strength of 1.2 V/cm.

Tolouee M., Hendriks K.D., Lie F.F., Gartzke L.P., Goris M., Hoogstra-Berends F., Bergink S, & Henning R.H. (2022). Cooling of Cells and Organs Confers Extensive DNA Strand Breaks Through Oxidative Stress and ATP Depletion. Cell Transplantation, 31, 09636897221108705.

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Detecting DNA Double-Strand Breaks

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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.

Piberger A.L., Bowry A., Kelly R.D., Walker A.K., González-Acosta D., Bailey L.J., Doherty A.J., Méndez J., Morris J.R., Bryant H.E, & Petermann E. (2020). PrimPol-dependent single-stranded gap formation mediates homologous recombination at bulky DNA adducts. Nature Communications, 11, 5863.

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Quantifying Chromosomal Double-Strand Breaks

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Cells (2 × 10⁶) 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⁻¹) at room temperature for 48 h and washed three times in TE buffer. Inserts were loaded onto a separation gel (1% chromosomal-grade agarose, Bio-Rad). 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 Mb) were quantified by densitometry using ImageJ. Intensity of DNA entering the gel was normalized to total DNA and untreated control to obtain final values.

Kotsantis P., Silva L.M., Irmscher S., Jones R.M., Folkes L., Gromak N, & Petermann E. (2016). Increased global transcription activity as a mechanism of replication stress in cancer. Nature Communications, 7, 13087.

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DNA Double-Strand Break Repair Kinetics

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The repair kinetics of DNA DSBs in early log-phase cells treated with 10 μg/ml Phl for 30 min were analyzed by PFGE. Plugs were prepared as described in the manufacture's instruction (CHEF Genomic DNA Plug Kits, Bio-Rad Laboratories, Inc., USA) with the following modifications: 5 × 10⁸ cells were washed twice in 30 ml of CSE buffer (20 mM citrate/phosphate [pH 5.6], 40 mM EDTA, 1.2 M sorbitol) and then incubated for 90 min at 37°C in 5 ml of CSE containing 1.5 mg/ml Zymolyase-20T (Seikagaku Corporation, Japan) for cell wall digestion. The cell pellet was then resuspended in 300 μl of TSE buffer (10 mM Tris–HCl [pH 7.5], 0.9 M sorbitol, 45 mM EDTA) and mixed with 400 μl of 1% low melting point agarose in TSE and dispensed in 100 μl aliquots to plugs molds. Cell lysis was performed by incubating gelled plugs in 0.25 M EDTA, 50 mM Tris–HCl [pH 7.5], 1% SDS for 90 min at 55°C, followed by two 24 h incubations in 1% lauryl sarcosine, 0.5 M EDTA [pH 9.5], and 1 mg/ml proteinase K at 55°C. Plugs were stored at 4°C in Tris–EDTA and washed three times in Tris–EDTA before loading. Pulse-field gel electrophoresis was carried out in 0.8% chromosomal grade agarose (Bio-Rad) in a Bio-Rad CHEF-DRII apparatus. Electrophoresis (Pulse time: 1800 s, 2 V/cm, angle: 100°) was carried out for 48 h at 14°C in TAE buffer. Finally, the agarose gel was stained in 0.5 μg/ml ethidium bromide for 30 min.

Manjón E., Edreira T., Muñoz S, & Sánchez Y. (2017). Rgf1p (Rho1p GEF) is required for double-strand break repair in fission yeast. Nucleic Acids Research, 45(9), 5269-5284.

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