Gel-based E. coli DNA Topoisomerase II (Gyrase) Assay Kit Plus-100 (for 100 assays)
BHE16300100
This product includes 400 ul of 10 x Buffer, 105 ul of 250 ug/ml relaxed DNA, 220 ul of 10 mM ATP, 550 ul of 5 x Gel loading buffer and 25 ul of 2000 nM E.coli gyrase.
DNA topoisomerases such as bacterial topoisomerase II (gyrase) convert relaxed circular DNA into supercoiled DNA (DNA supercoiling reaction). The Gel-Based E. coli Topoisomerase II (Gyrase) DNA Supercoiling Assay Kit is based on the principle that the supercoiled DNA and relaxed DNA are separated by agarose gel electrophoresis. Fluorescence-based DNA supercoiling assays in a 96-well pate format are also available for high throughput screening of gyrase inhibitors. For more information of the high throughput gyrase DNA supercoiling assays, please visit the website at http://www.profoldin.com/topoisomerase_assays_1.html.
The Gel-Based E. coli DNA Topoisomerase II (Gyrase) Assay Kit Plus-100 (Catalog No. GDSA100KE) includes all the reagents for 100 samples in gel-based assays of E. coli gyrase DNA supercoiling activity. It includes 400 µl of 10 x Buffer, 105 µl of 250µg/ml relaxed DNA, 220 µl of 10 mM ATP, 550 µl of 5 x Gel loading buffer and 25 µl of 2000 nM E.coli gyrase (100 x).
Assay Protocol
1. Reaction:
The total volume of each reaction mixture is 20µl including: 13µl of H2O, 2µl of 10 x Buffer, 1µl of 250µg/ml relaxed DNA, 2µl of 200 nM E. coli gyrase (10 x), 2µl of 10 mM ATP. Incubate the reaction mixture at 37°C for 60 min. At the end of the reaction, add 5µl of 5 x gel loading buffer.
Note: The final concentrations are 20 mM Tris-HCl, pH 8, 35 mM NH4OAc, 4.6 % glycerol, 1 mM DTT, 0.005% Brij35, 8 mM MgCl2, 12.5µg/ml relaxed plasmid DNA, 1 mM ATP and 20 nM topoisomerase II. The 10 x enzyme is prepared by dilution of the 100 x enzyme in 1x assay buffer. A negative control reaction can be the reaction mixture without addition of ATP.
2. Agarose gel electrophoresis
(1) Prepare 1 % agarose gel in 1 x TAE buffer.
(2) Load 25 ul of the sample.
(3) Run the gel at 100 V for 90 min to 2 hours.
(4) Stain the gel in an ethidium bromide solution and destain the gel in water.
(5) Take a picture of the gel under UV light.
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