Deoxyribonuclease I is isolated from bovine pancreas and is processed to reduce RNase activity to below detectable levels.
DNase I digests single- and double-stranded DNA to a mixture of mono- and oligonucleotides carrying 5'-phosphates and 3'-OH termini. This catalytic activity is divalent ion-dependent. In the presence of Mg2+, DNase I hydrolyzes each strand of double-stranded DNA randomly and independently. In the presence of Mn2+, both strands can be cleaved. It is useful for nick translation, DNase footprinting, bisulfite-mediated mutagenesis, and RNA purification1,2, and is suitable for all but the most stringent RNA purification procedures.
However, RNA treated with this DNase I should not be used to generate a cDNA library or used for RT-PCR. For these sensitive applications, it is recommended to use DNase I, Amplification Grade, Catalog Number AMPD1.
Deoxyribonuclease I is supplied as a solution in 50% (v/v) glycerol, 20 mM sodium acetate, pH 6.5, 5 mMCaCl2, 0.1 mM PMSF.
Specific activity: ≥10,000 units/mg protein
Unit Definition: One unit will cause a ΔA260 of 0.001 per minute per mL reaction mixture using calf thymus DNA as substrate.
Typical protocol to remove trace DNA
Add to an RNase-free PCR tube:
1 µg of RNA sample in 8 µL water
1 µL of 10X reaction buffer (200 mM Tris-HCl, pH 8.3, with 20 mM MgCl2)
1 µL of DNase I, 1 unit/µL*
*Refer to the Certificate of Analysis for the lot specific specific activity and the number of mg protein per mL.
Incubate for 15 minutes at room temperature
To stop add 1 µL of Stop Solution to bind calcium and magnesium ions and to inactivate the DNase I.
The Stop Solution (50 mM EDTA) must be added before heating to prevent metal (Mg/Ca) ion catalyzed hydrolysis of the RNA. Heat at 70 °C for 10 minutes to denature both the DNase I and the RNA.
Note: This product should not be used for digestions longer than 15 minutes or for digestions at temperatures higher than room temperature, or the residual contaminating RNase activity will begin to degrade the RNA.
Assay Buffer: 100 mM sodium acetate, pH 5.0 at 25 °C, containing 5 mM MgCl2
50 mg/µL calf thymus DNA
5-20 µg of DNase I was added to 3 mL of reaction mixture at 25 °C and the ΔA260 is monitored for 10 minutes. The maximum linear rate was used to calculate the activity.
Two µg transfer RNA were incubated with 2 µg DNase I in a 50 µL reaction mixture containing 30 mM Tris-HCl, pH 7.8, 50 mM NaCl and 10 mM MgCl2 for 16 hours at 37 °C. No degradation of the tRNA was detected by polyacrylamide gel