New England Biolabs, R0545S, BcgI

BcgI has been reformulated with Recombinant Albumin (rAlbumin) beginning with Lot #10271508. Related Categories Restriction Endonucleases B Applications 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™ 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: 75% NEBuffer™ r3.1: 100% rCutSmart™ Buffer: 50% Diluent Compatibility Diluent A Storage Buffer 10 mM Tris-HCl 50 mM KCl 1 mM DTT 0.1 mM EDTA 200 µg/ml Recombinant Albumin 0.32 mM S-adenosylmethionine (SAM) 50% Glycerol pH 7.4 @ 25°C Heat Inactivation 65°C for 20 minutes Methylation Sensitivity dam methylation: Impaired by Overlapping dcm methylation: Not Sensitive CpG Methylation: Blocked by Some Combinations of Overlapping FAQ 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: Do BcgI generated ends ligate? A: BcgI excises a 32 bp fragment with 2-base 3´ overhangs. These overhangs are not specific, so whether the ends can be ligated depends entirely on the specific DNA sequence being cut. Q: Is this enzyme sensitive to dam, dcm or mammalian CpG methylation? A: Yes. This enzyme cannot cut recognition sites that are impaired by overlapping dam methylation or blocked by some combinations of overlapping 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.