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Gel-Based Human Topoisomerase I DNA relaxation Assay Kit

BHA16300007

This product includes 400 ul of 10 x Buffer HT1, 100 ul of 0.25 mg/ml supercoiled DNA, 550 ul of 5 x Gel loading buffer and 10 ul of 500 U/ul human topoisomerase I enzyme.

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+1 866.986.9598
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[email protected]
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Product Manual Datasheet
Protocols Description Assay Protocol
1. Reaction: The total volume of each reaction mixture is 20 µl in an eppendorf tube including: 16 µl of H2O, 2 µl of 10 x Buffer HT1, 1 µl of 250 µg/ml supercoiled DNA, 1 µl of 50 U/µl human topo I. Incubate the reaction mixture at 37°C for 60 min. At the end of the reaction, add 5 µl of 5 x gel loading buffer.
 

Note: The final concentrations are 25 mM Tris-Cl, pH 7.4, 57 mM KCl, 5 mM MgCl2, 0.25 mM DTT, 0.25 mM EDTA and 15 µg/ml BSA, 12.5 µg/ml supercoiled plasmid DNA and 2.5 U/µl human topo I. The 50 U/µl enzyme solution is prepared freshly by dilution of the 500 U/µl stock solution in 1x assay buffer. The diluted enzyme may not be stable for storage. For inhibitor IC50 measurements, assay linearity should be tested to determine the optimal enzyme amount for the assay.
 
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.
SKU Supplier No. Size Your price Quantity
BHA16300007 GHRA100KE 100 assays $518.71
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