Elimination of template DNA following in vitro synthesis of RNA with T7, SP6, or T3 RNA polymerase.
Labeling of DNA by nick translation, in combination with Klenow or other DNA polymerases.
Treatment of RNA prior to RT-PCR.1
Characterization of DNA-protein interactions by DNase I footprinting.2,3
RNase-Free DNase I (bovine pancreas) is an endonuclease useful in removing DNA that might interfere with the characterization, manipulation, or use of RNA, or for any application requiring highly purified DNase I, such as nick translation. It efficiently hydrolyzes dsDNA and ssDNA into a mixture of short oligonucleotides and mononucleotides.
Figure 1. DNA removal from in vitro transcription reactions using RNase-Free DNase I. A linearized DNA template was transcribed using T7 RNA polymerase according to standardin vitro transcription conditions. Lane 1, kb ladder; Lane 2, DNA control; Lane 3, transcription mixture; Lane 4, transcription mixture treated with 1 MBU of RNase-Free DNase I for 15 minutes at 37°C.
Unit Definition: One Molecular Biology Unit (MBU) of RNase-Free DNase I digests 1 µg of pUC19 DNA to oligodeoxynucleotides in 10 minutes at 37°C under standard assay conditions. Storage Buffer: 50% glycerol containing 10 mM Tris-HCl (pH 7.5), 10 mM CaCl2, and 10 mM MgCl2. DNase I 10X Reaction Buffer: 100 mM Tris-HCl (pH 7.5), 25 mM MgCl2, and 5 mM CaCl2. Quality Control: No degradation of 1 µg of a synthetic RNA transcript is detected by agarose gel electrophoresis following incubation with 10 U of RNase-Free DNase I at 37°C for 1 hour. References
Kienzle, N. et al. (1996) BioTechniques 20, 612.
Sambrook, J. et al. (1989) in: Molecular Cloning: A Laboratory Manual (2nd ed.) Cold Spring Harbor Laboratory Press, New York.
Galas, D.J. and Schmitz, A. (1978) Nucleic Acids Res. 5, 3157.
