Description: E. coli DNA gyrase were purified from E. coli strain BL21(DE3) carrying plasmids overexpressing His-tagged subunit A (GyrA) and subunit B (GyrB), respectively. After the His-tag was removed, the DNA gyrase was assembled at an equal ratio of GyrA and GyrB to a tetrameric holoenzyme: GyrA2GyrB2. A 12% SDS PAGE gel shows the purity of E. coli DNA gyrase. E. coli DNA gyrase is stored in the storage or dilution buffer: 50 mM Tris-HCl, pH 8, 100 mM KCl, 2 mM DTT, 0.1 mg/mL BSA, and 10% glycerol. E. coli DNA gyrase is free from contaminations by other E. coli DNA topoisomerases including DNA topoisomerase IV. E. coli DNA gyrase is also free from DNase and RNase contaminations.
Unit definition: One unit of E. coli DNA gyrase is defined to completely supercoil 200 ng of relaxed plasmid pAB1 in 30 μL of 1xDNA gyrase buffer (35 mM Tris-HCl, pH 8, 24 mM KCl, 4 mM MgCl2, 2 mM ATP, 2 mM DTT, 0.1 mg/mL BSA, and 5% glycerol) at 37 °C in 15 min via agarose gel-based assays and fluorescence-based assays as well.
Concentrations: Two concentrations of E. coli DNA gyrase are available: 1,000 units/mL (0.1 mg/mL and 270 nM) and 10,000 units/mL (1 mg/mL and 2.7 μM). It is recommended that the enzyme of 1,000 units/mL should be used as is and not be diluted. Please contact us for higher concentrations of E. coli DNA gyrase and gyrase without BSA.
Storage and shipping: E. coli DNA gyrase is shipped and should be stored in a -80°C freezer (-70-80°C) and is stable for at least 12 months in this condition. E. coli DNA gyrase can be aliquoted on first thawing to prevent loss of activities from freeze-thaw cycles.
Dilution buffer: 50 mM Tris-HCl, pH 8, 100 mM KCl, 2 mM DTT, 0.1 mg/mL BSA, and 10% glycerol. Dilution buffer is supplied.
5xDNA gyrase assay buffer: 175 mM Tris-HCl, pH 8, 120 mM KCl, 20 mM MgCl2, 10 mM ATP, 10 mM DTT, 0.5 mg/mL BSA, and 25% glycerol. This assay buffer does not contain spermidine. 5xDNA gyrase buffer is supplied.
Stop solution: 250 mM EDTA and 3% SDS.
Unit definition: One unit of E. coli DNA gyrase is defined to completely supercoil 200 ng of relaxed plasmid pAB1 in 30 μL of 1xDNA gyrase buffer (35 mM Tris-HCl, pH 8, 24 mM KCl, 4 mM MgCl2, 2 mM ATP, 2 mM DTT, 0.1 mg/mL BSA, and 5% glycerol) at 37 °C in 15 min via agarose gel-based assays and fluorescence-based assays as well.
Concentrations: Two concentrations of E. coli DNA gyrase are available: 1,000 units/mL (0.1 mg/mL and 270 nM) and 10,000 units/mL (1 mg/mL and 2.7 μM). It is recommended that the enzyme of 1,000 units/mL should be used as is and not be diluted. Please contact us for higher concentrations of E. coli DNA gyrase and gyrase without BSA.
Storage and shipping: E. coli DNA gyrase is shipped and should be stored in a -80°C freezer (-70-80°C) and is stable for at least 12 months in this condition. E. coli DNA gyrase can be aliquoted on first thawing to prevent loss of activities from freeze-thaw cycles.
Dilution buffer: 50 mM Tris-HCl, pH 8, 100 mM KCl, 2 mM DTT, 0.1 mg/mL BSA, and 10% glycerol. Dilution buffer is supplied.
5xDNA gyrase assay buffer: 175 mM Tris-HCl, pH 8, 120 mM KCl, 20 mM MgCl2, 10 mM ATP, 10 mM DTT, 0.5 mg/mL BSA, and 25% glycerol. This assay buffer does not contain spermidine. 5xDNA gyrase buffer is supplied.
Stop solution: 250 mM EDTA and 3% SDS.
This product is used for research only.
DNA Supercoiling Assay Instruction: