New England Biolabs, R0502S, BspMI

BspMI has been reformulated with Recombinant Albumin (rAlbumin) beginning with Lot #10271507. 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: 50% NEBuffer™ r3.1: 100% rCutSmart™ Buffer: 10% Diluent Compatibility Diluent B Storage Buffer 10 mM Tris-HCl 500 mM NaCl 1 mM DTT 0.1 mM EDTA 200 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: What is Star Activity and how can it be avoided? A: It has been demonstrated that under extreme non-standard conditions, restriction endonucleases are capable of cleaving sequences which are similar but not identical to their defined recognition sequence. This altered or relaxed specificity has been termed ""star"" activity. It has been suggested that star activity may be a general property of restriction endonucleases and that any restriction endonuclease can be made to cleave noncanonical sites under certain extreme conditions. The manner in which an enzyme's specificity is altered depends on the enzyme and on the conditions employed to induce the star activity. The most common types of altered activity are single base substitutions, truncation of the outer bases in the recognition sequence, and single-strand nicking. Star activity is completely controllable in the vast majority of cases and is generally not a concern when performing restriction endonuclease digests. New England Biolabs' enzymes will not exhibit star activity when used under recommended conditions in their supplied NEBuffers. Listed below are reaction conditions known to induce or inhibit star activity. Conditions that Contribute to Star Activity 1. High glycerol concentration [> 5% v/v] 2. High units to µg of DNA ratio [Varies with each enzyme, usually >100 units/µg] 3. Low ionic strength [< 25 mM] 4. High pH [> pH 8.0] 5. Presence of organic solvents [DMSO, ethanol, ethylene glycol, dimethylacetamide, dimethylformamide, sulphalane] 6. Substitution of Mg++ with other divalent cations [Mn++, Cu++, Co++, Zn++] Inhibiting Star Activity If you are concerned about star activity, we recommend the following guidelines. 1. Use as few units as possible to get a complete digestion. This avoids overdigestion and reduces the final glycerol concentration in the reaction. 2. Make sure the reaction is free of any organic solvents such as alcohols which might be present in the DNA preparation. 3. Raise the ionic strength of the reaction buffer to 100-150 mM (provided the enzyme is not inhibited by high salt). 4. Lower the pH of the reaction buffer to pH 7.0. 5. Use Mg++ as the divalent cation. 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.