Deoxyribonuclease II - Assay
Deoxyribonuclease II Assay
- 1.0 M Acetic acid, pH 4.6
- 0.02 M Magnesium sulfate
Substrate: Dilute 12.5 ml of 0.02 M magnesium sulfate to a volume of 200 ml with reagent grade water. Add 10 mg Worthington calf thymus DNA (Code: DNA). Let stand overnight at room temperature. Add 25 ml of 1.0 M acetic acid, pH 4.6 and adjust the pH to 4.6. Dilute to a final volume of 250 ml with reagent grade water. Store at 2 - 8°C.
Dissolve enzyme at one mg/ml in reagent grade water. Dilute further in water immediately prior to use to a concentration of 50 - 150 units/ml.
Adjust the spectrophotometer to 260 nm and 25°C.
Pipette 2.5 ml of substrate into cuvette and incubate in the spectrophotometer for 3 - 4 minutes to establish blank rate, if any, and to reach temperature equilibrium. Add 0.5 ml diluted enzyme. Record increase in A260 from linear portion of curve. Note: the change in A260 for this assay is not generally linear from the initial time and is linear for only short periods. The change in A260 should be determined from the portion which is most linear.