DNase I Recombinant, RNase-free Protocol

Product No. 04716728001

Protocol

REMOVAL OF DNA FROM RT-PCR REACTIONS:

Overview

DNase I from bovine pancreas is a glycoprotein of Mr 37000. A special procedure is used to remove RNases from the DNase preparation. DNase I is a DNA-specific endonuclease that hydrolyzes ds or ssDNA to a mixture of oligo and mononucleotides. The enzyme requires divalent cations for maximal activity. The specificity of the reaction depends on the nature of the cations. In the presence of Mg²⁺ DNase I cause nicks in dsDNA, while in the presence of Mn²⁺ the enzyme produces double-stranded breaks.

Suggested Method for the Removal of DNA for RT-PCR

10 x reaction buffer: 200 mM Tris-HCl, pH 8.3 500 mM KCl 10 mM MnCl₂ recommended to add 20 U Protector RNase Inhibitor;
Add 1 U DNase I, RNase-free per 1 μg total RNA and incubate for 30 min at +25 °C.

Stop the reaction by phenol/chloroform extraction and subsequent ethanol precipitation (alternatively, stop the reaction by heating for 5 min at +75 °C, but be aware that increased temperatures can damage RNA).

Note: one-tube RT-PCR

Removal of Mn²⁺ buffer and enzyme is necessary to avoid disturbances in one step RT-PCR with the Titan-One-Tube RT-PCR system. In this case, an alternative method can be used:

1. Dissolve 1 μg RNA in 40 mM Tris, pH 8, 10 mM MgSO₄, 1mM CaCl₂
2. Add 6 U DNase I, RNase-free and 20 U Protector RNAse Inhibitor.
3. Incubate for 1 h at +37 °C

Inactivate for 5 min at +90 °C, spin down by short centrifugation and incubate on ice immediately