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Reaction buffer:50 mM Tris-HCl, 1 mM MgCl2, pH 8.0 （In the case of extensive dilution before use, carrier protein such as 0.1 mg/ml HSA or BSA is generally recommended to avoid any enzyme loss from surface adsorption)DNA Substrate: 1 mg/ml salmon sperm DNA is dissolved overnight at 4 ℃, in reaction buffer, and is then sonicated on ice to obtain a homogenous solution.Enzyme: Different dilution of nuclease with reaction bufferStop reagent: Trichloroacetic acid (TCA)
Measurement of activity: The activity of any unknown nuclease can be determined from a single measurement by means of the standard curve. The specific activity of BenzNuclease is >1.5 x 10 e6 unit/mg protein.
Standard curve establishment:- 400 μl substrate + 100 μl enzyme of known activity = 500 μl mixture- Incubate the mixture at 37℃ for 30 min.- Stop the reaction by addition of 400 μl cold TCA and incubate on ice for 10 min.- Centrifuge at 8500 g for 5 min.- Measure the absorbance of supernatant at 260 nm.- Lot a standard curve with nuclease of known activities for each set of measurements.