New England Biolabs, R0559L, BsgI

BsgI has been reformulated with Recombinant Albumin (rAlbumin) beginning with Lot #10201941. 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 volume of 50 μl. Reaction Conditions 1X rCutSmart™ Buffer Incubate at 37°C 1X rCutSmart™ Buffer 50 mM Potassium Acetate 20 mM Tris-acetate 10 mM Magnesium Acetate 100 µg/ml Recombinant Albumin (pH 7.9 @ 25°C) Activity in NEBuffers NEBuffer™ r1.1: 25% NEBuffer™ r2.1: 50% NEBuffer™ r3.1: 25% rCutSmart™ Buffer: 100% Diluent Compatibility Diluent B Storage Buffer 300 mM NaCl 10 mM Tris-HCl 1 mM DTT 0.1 mM EDTA 0.32 mM S-adenosylmethionine (SAM) 500 µg/ml Recombinant Albumin 50% Glycerol pH 7.4 @ 25°C Heat Inactivation 65°C for 20 minutes Methylation Sensitivity dam methylation: Not Sensitive dcm methylation: Not Sensitive CpG Methylation: Not Sensitive FAQ Q: Why doesn't BsgI cut well? A: BsgI requires 80 µM SAM in the reaction mixture. Incubation without SAM results in 25-50% activity. SAM should be fresh. We suggest making up the SAM just before usage and even aliquoting it out in the concentrated form so it does not have to undergo too many freeze-thaws. Even at -70°C it only lasts about six months. BsgI and SAM lose activity in overnight digests, so they are not recommended. It is better to add more units of BsgI and to spike once with fresh SAM than to do an overnight digest. BsgI is also inhibited by nucleotides and DNA contaminants. Drop dialysis of the DNA can increase cleavage. Q: What is the activity of BsgI at 25°C? A: BsgI is 100% active at 25°C. Q: Are there known sequence errors at the recognition site in a database? A: Yes. BsgI cuts phiX174 DNA; reports of no BsgI site in that DNA have been attributed to a sequencing error. 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: 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: 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: Does BsgI require SAM for activity? A: Yes, S-adenosylmethionine (SAM) is required for optimal activity (SAM is supplied in the enzyme formulation as of October 2020). If you are unsure whether SAM is in the enzyme formulation and you have a vial of SAM from NEB, it is fine to add SAM to the reaction per previous guidelines. 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.