New England Biolabs, B0570S, DNase I-XT Reaction Buffer

Related Categories Exonucleases and Non-specific Endonucleases,, Buffers Specification 1X Buffer Components 10 mM Tris-HCl 50 mM NaCl 10 mM MgCl2 0.5 mM CaCl2 0.005% Tween® 20 pH 7.6@25°C FAQ Q: What is the difference between DNase I (NEB #M0303) and DNase I-XT (NEB #M0570)? A: DNase I is inhibited by salt concentrations >50 mM. DNase I-XT is a salt-tolerant DNase I that retains activity up to 300 mM salt but has optimal activity at salt concentrations between 50-100 mM. DNase I-XT is, therefore, the preferred enzyme for DNA template removal from an in vitro transcription (IVT) reaction. Q: Will DNase I-XT (NEB #M0570) work in NEB buffers 1-4? A: Yes, but not as efficiently as it will in the DNase I-XT Reaction Buffer (NEB #B0570). DNase I-XT is a calcium-dependent enzyme that works best in DNase I-XT Reaction Buffer, which contains calcium chloride. It is recommended that even if you are using NEB buffers 1-4 you also add DNase I-XT buffer or 0.5 mM CaCl2 to the reaction. Q: What is the best way to remove DNase I-XT from my reaction? A: The best way to remove DNase I-XT from your reaction is to perform a phenol/chloroform extraction or to use a spin column. Heat inactivation is not recommended as it may not completely inactivate all of the DNase I-XT, and it could interfere with your downstream applications. Q: Will DNase I-XT work in rCutSmart buffer? A: Yes, DNase I-XT will work in rCutSmart Buffer, with the addition of calcium (0.5 mM CaCl2). 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® RNA Cleanup Kit (NEB #T2030, #T2040, #T2050) to cleanup up my DNase I-XT (NEB #M0570)-treated RNA? A: Yes, the Monarch RNA Cleanup Kits will remove residual DNase I-XT activity from RNA samples. On-column treatment with DNase I/DNase I-XT can often result in residual DNase I activity, therefore, we do not recommend on-column DNase I/DNase I-XT treatment with the Monarch RNA Cleanup Kit, but rather in-solution DNase I /DNase I-XT treatment prior to RNA cleanup.