New England Biolabs, M0303L, DNase I (RNase-free)

Related Categories Chromatin Analysis,, Exonucleases and Non-specific Endonucleases,, RNA Synthesis In vitro Transcription (IVT), Applications RNA Cloning Specification Materials Required but not Supplied Nuclease-free Water (NEB #B1500) RNase Inhibitor, Murine (NEB #M0314) EDTA Unit Definition One unit is defined as the amount of enzyme which will completely degrade 1 µg of pBR322 DNA in 10 minutes at 37°C in DNase I Reaction Buffer. Complete degradation is defined as the reduction of the majority of DNA fragments to tetranucleotides or smaller. Reaction Conditions 1X DNase I Reaction Buffer Incubate at 37°C 1X DNase I Reaction Buffer 10 mM Tris-HCl 2.5 mM MgCl2 0.5 mM CaCl2 (pH 7.6 @ 25°C) Usage Concentration 2,000 units/ml Storage Buffer 10 mM Tris-HCl 2 mM CaCl2 50% Glycerol pH 7.6 @ 25°C Heat Inactivation 75°C for 10 minutes FAQ Q: What is the specific activity of DNase I(RNase-free)? A: The specific activity is approximately 20,000 Units/mg. Q: Will DNase I work in NEB buffers 1-4? A: Yes, but not as efficiently. DNase I is a Calcium dependent enzyme so it works best in it’s own buffer which contains Calcium Chloride. It is recommended that even if you are using NEB buffers 1-4 that you also add DNase I buffer or .5mM CaCl2 to the reaction. Also, DNase I does not like high salt, as contained in NEB buffer 3. It works least efficiently in buffer 3, but will still give 50-75% digestion. Q: What is the best way to remove DNase I from my reaction? A: The best way to remove DNase I from your reaction is to perform a phenol/chloroform extraction or to use a spin column. You can do the heat inactivation step, but that may not completely remove all of the DNase I, and it could interfere with your downstream applications. Q: Will DNase I work in rCutSmart buffer? A: Yes, DNase I will work in rCutSmart, with the addition of calcium. To see its % functional activity in rCutSmart, and that of other DNA Modifying enzymes in the cloning workflow, refer to the Activity of DNA Modifying Enzymes in rCutSmart® Buffer chart. Q: Can I use the Monarch Spin RNA Cleanup Kit to cleanup up my DNase I-treated RNA? A: Yes, the Monarch Spin RNA Cleanup Kits will remove residual DNase I (NEB #M0303) activity from RNA samples. On-column treatment with DNase I can often result in residual DNase I activity, therefore, we do not recommend on-column DNase I treatment with the Monarch Spin RNA Cleanup Kit, but rather in-solution DNase I treatment prior to RNA cleanup.