Calf thymus DNA

Nuclease activity assay [1]:

1. Solution preparation: Calf thymus DNA is dissolved in sterile ultrapure water (without DNase I) to prepare a 1mg/mL solution. During the dissolution process, it can be gently inverted at 4°C to aid dissolution, avoiding ultrasonic and vigorous vibration. After overnight incubation at 4°C, it can be fully dissolved;

Note: When the concentration is low (25μg/mL), DNA is prone to adsorb to the inner wall of the plastic tube. DNA is easily degraded in solutions with a pH greater than 8.0, so it cannot be dissolved in alkaline solutions.

2. Negative control group: 7.5 μg (7.5μL from the 1mg/mL solution) of Calf thymus DNA is mixed with 50μL of Tris;Positive control group: 7.5μg (7.5μL from the 1mg/mL solution) of Calf thymus DNA is mixed with 40μL of Tris and 10μL of DNase I (2U/mL);

3. All samples are incubated at 37°C for 6 hours;

4. After incubation, the complete DNA is precipitated with phenol-chloroform;

5. DNA is visualized on a 1% agarose gel containing Roti-Gel Stain;

6. The relative nuclease activity is determined by comparing with standard nuclease activities (0, 0.015, 0.03, 0.06, 0.125, 0.25, 0.5, and 1U/mL of DNase I).

This protocol only provides a guideline, and should be modified according to your specific needs.

References:
[1] Fingerhut L, Ohnesorge B, von Borstel M, et al. Neutrophil Extracellular Traps in the Pathogenesis of Equine Recurrent Uveitis (ERU). Cells. 2019;8(12):1528. Published 2019 Nov 27.