New England Biolabs, R0681T, Nb.BssSI

Related Categories Nicking Endonucleases,, Restriction Endonucleases: N-O Specification Unit Definition One unit is defined as the amount of enzyme required to convert 1 μg of supercoiled pUC19 DNA to open circular form in 1 hour at 37°C in a total reaction volume of 50 μl. Reaction Conditions 1X NEBuffer™ r3.1 Incubate at 37°C 1X NEBuffer™ r3.1 100 mM NaCl 50 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: 100% rCutSmart™ Buffer: 25% Diluent Compatibility Diluent B Storage Buffer 300 mM NaCl 10 mM Tris-HCl 1 mM DTT 0.1 mM EDTA 500 µg/ml BSA 50% Glycerol pH 7.4 @ 25°C Heat Inactivation No Methylation Sensitivity dam methylation: Not Sensitive dcm methylation: Not Sensitive CpG Methylation: Not Sensitive FAQ Q: Is Nb.BssSI active at higher temperatures? A: Nb.BssSI is active at 50°C and exhibits ~3 fold higher activity at this temperature. Q: How should I stop my nicking reaction if enzyme cannot be heat inactivated? A: If no further manipulations of the digested DNA are planned, the reaction can be terminated by adding a stop solution. At NEB, we use the following stop solution: 50% glycerol, 50 mM EDTA (pH 8.0), and 0.05% bromophenol blue (10 μl / 50 μl reaction). Phenol/chloroform extraction is another means of inactivating a restriction enzyme. Q: I tested your restriction enzyme on the substrate DNA recommended by NEB, and it appears to be active, however it does not digest my DNA. What could be the reason? A: The quality and cleanliness of the DNA are major factors that can affect enzyme activity. Restriction Enzymes active in rCutSmart® Buffer, but not as active in NEBuffer r2.1 and/or r3.1 (our higher salt buffers), can be inhibited by salt in the reaction. DNA purification procedures that use spin columns can result in DNA solutions with significant levels of salt that can carry over into the reaction and inhibit enzyme activity. To prevent this, we recommend that the DNA solution added to the reaction be no more than 25% of the total reaction volume. This can be attained by adjusting the total reaction volume accordingly. Q: Are there any nicking enzymes that are used on the Irys® system from BioNano Genomics®? A: Nb.BssSI and Nt.BspQI are nicking endonucleases that cleave only one strand of DNA on a double-stranded DNA substrate. They can be used for genome mapping, assembly and SV detection on the Irys system from BioNano Genomics. For more information please visit www.BioNanogenomics.com or contact BioNano at : support@bionanogenomics.com Q: What is the activity of nicking enzymes at different temperatures? A: The activity of NEB nicking enzymes at different temperatures can be found on the table "Effect of Various Temperatures on Nicking Endonucleases”. Q: Can I purchase large amounts of an existing Enzyme for Innovation? A: Yes, if you have use for large amounts of an Enzyme for Innovation, please contact NEB at EnzymesForInnovation@neb.com. Please note that there may be limited availability and thus extended lead times required for large orders. Additional QC testing may also be necessary to meet your specific application and needs. Q: What are Enzymes for Innovation? A: Enzymes for Innovation (EFI) is a project initiated by NEB to provide unique enzymes to the scientific community in the hopes of enabling the discovery of new and innovative applications. These enzymes have interesting properties and unique specificities that are not commercially available elsewhere at the quality that you would expect from NEB. Enzymes for Innovation graduates are enzymes that have transitioned from having unknown or exploratory applications to becoming the cornerstone of a defined application. If you have an idea for an “Enzyme for Innovation” with a suggested application that may be useful, please email us at EnzymesForInnovation@neb.com. For more information, please view video. 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.