Ultra-active Nuclease (EC: 3.1.30.2), also known as Benzonase, cleaves all single and/or double stranded DNA and RNA in both linear and/or circular forms under a wide range of cleavage conditions. It is a recombination endonuclease expressed and purified from E Coli which is a non-covalent homo-dimer. Each single chain has 245 amino acids with apparent molecular weight of ~29 kD on the reduced SDS-PAGE gel. This product is mainly used for the removal of residual nucleic acid DNA/RNA contaminates from biological expression systems for proteins, vaccines, and antibodies and reduces viscosity of the cell lysates in the downstream purification. The efficiency may vary with buffers, cell types and cell lysis methods. Typically 200-500 units of Benzonase are used per gram of cell paste. Add Benzonase before lysis. Incubate the mixture with shaking or rotating gently for 10-20 min at the room temperature.
Applications:
Technical notes:
Documentation for Assays:
1. SDS-PAGE GEL of Benzonase under both reduced & non-reduced conditions
Applications:
- Benzonase is ideal for a wide variety of applications where complete digestion of DNA/RNA is desirable
- Removal of nucleic acids DNA/RNA from target proteins in tissue or cell lysates
- Sample preparation in western blotting or two- dimensional gel electrophoresis
- Viscosity reduction in protein purification from extracts
Technical notes:
- Purity ≥99% by Coomassie Blue Staining
- Specific activity ≥1 X 106 U/mg or ≥1 X 106 U/ml
- Endotoxin ≤ 0.2 EU/ml (0.0001 EU/ 1000 U)
- Unit Definition One unit is defined as the amount of enzyme that causes a ∆A260 of 1.0 in 30 min, which corresponds to complete digestion of 37 μg of DNA
- Standard reaction conditions are 1 mg/ml sonicated DNA substrate in the buffer of 50 mM Tris▪HCl at pH 8.0 with 0.1 mg/ml BSA and 1 mM MgCl2, incubated at 37 °C
- Storage and Shipping Stored in the buffer of 50 mM Tris-HCl at pH 8 with 50 mM NaCl and 2 mM MgCl2. The enzyme is stable for at least one year when stored at -20 °C
Documentation for Assays:
1. SDS-PAGE GEL of Benzonase under both reduced & non-reduced conditions
SDS-PAGE gel Lanes 1-3 under the reduced condition: 1.0, 2.0 and 5 μg of Ultra-active Nuclease loaded, respectively and Lanes 4-6 under the non-reduced condition: 5.0, 2.0 and 1.0 μg of Ultra-active Nuclease loaded, respectively. Stained with Coomassie Blue.
2. Actual cleavage of DNA plasmid with benzonase and visualized by gel
The activity was confirmed by actual cleavage of 37 ug DNA plasmid with 0, 1/10 U, 1/4 U, 1/2 U, 3/4 U and 1 U from lanes 1 to 6 at 37 C for 30 minutes and visualized by 1.5 % agarose gel with ethidium bromide staining. The DNA plasmid dismissed gradually as benzonase was increased. The dramatic change occurred from 1/2 U to 1U (from lanes 4 to 6). The DNA plasmid was completely disappeared in lane 6 under 1 U of benzonase cleavage.