New England Biolabs, R0574S, BsrDI

New 37°C incubation temperature. Related Categories Restriction Endonucleases B,, Time-Saver Qualified Restriction Enzymes Applications Fast Cloning: Accelerate your cloning workflows with reagents from NEB,, Restriction Enzyme Digestion Specification Unit Definition One unit is defined as the amount of enzyme required to digest 1 µg of λ DNA in 1 hour at 37°C in a total reaction volume of 50 μl. Reaction Conditions 1X NEBuffer™ r2.1 Incubate at 37°C 1X NEBuffer™ r2.1 50 mM NaCl 10 mM Tris-HCl 10 mM MgCl2 100 µg/ml Recombinant Albumin (pH 7.9 @ 25°C) Activity in NEBuffers NEBuffer™ r1.1: 10% NEBuffer™ r2.1: 100% NEBuffer™ r3.1: 75% rCutSmart™ Buffer: 25% Diluent Compatibility Diluent A Storage Buffer 10 mM Tris-HCl 100 mM NaCl 1 mM DTT 0.1 mM EDTA 200 µg/ml Recombinant Albumin 50% Glycerol pH 7.4 @ 25°C Heat Inactivation 80°C for 20 minutes Methylation Sensitivity dam methylation: Not Sensitive dcm methylation: Not Sensitive CpG Methylation: Not Sensitive Activity at Temperature @65°C: 100% FAQ Q: What is the activity of BsrDI at 25°C? A: BsrDI is approximately 10% active at 25°C. Q: Are there any single BsrDI sites in common vectors? A: Yes, pACYC184 has a single BsrDI site. Q: Do I have to set-up digests with Time-Saver™ qualified enzymes for 5-15 minutes? Can I digest longer? A: NEB's Time-Saver™ enzymes have the benefit of working fast (5-15 minutes), but are also designed and qualified to withstand overnight digestions without degradation of DNA. Q: Is this enzyme sensitive to dam, dcm or mammalian CpG methylation? A: No. This enzyme is not sensitive to dam, dcm, or mammalian CpG methylation. For up-to-date information about methylation sensitivities, please visit Dam-Dcm and CpG Methylation or REBASE. Q: Can you tell me more about the switch from BSA to Recombinant Albumin (rAlbumin) in NEBuffers? A: NEB is excited to announce that we have switched BSA-containing reaction buffers (NEBuffer 1.1, 2.1, 3.1 and CutSmart® Buffer) to Recombinant Albumin-containing buffers (NEBuffer r1.1, r2.1, r3.1 and rCutSmart™ Buffer). We are also in the process of moving all restriction enzyme formulations to contain rAlbumin. We feel that moving away from animal-containing products is a step in the right direction and are able to offer this enhancement at the same price. Q: In an effort to standardize reaction conditions, the recommended incubation temperature has changed for ApaI (NEB #R0114), BaeI (NEB #R0613), BsrDI (NEB #R0574) and SmaI (NEB #R0141). Which incubation temperature should I use? A: You can safely incubate at either temperature. Please note that for SmaI, we suggest incubating at 25°C if you are doing extended digestions (>1 hour). Q: Are certain restriction enzymes more active at higher incubation temperatures than what is recommended? A: NEB restriction enzymes are 100% active at the recommended incubation temperature in the recommended buffer provided with each enzyme. With some restriction enzymes isolated from thermophilic species, you may see increased activity at higher incubation temperatures. We do not recommend increasing the temperature as additional nuclease activities may appear at non-recommended temperatures.