New England Biolabs, R0599L, Acc65I

Related Categories Restriction Endonucleases: A,, 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 pBC4 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: 25% Diluent Compatibility Diluent A Storage Buffer 10 mM Tris-HCl 100 mM NaCl 1 mM DTT 0.1 mM EDTA 200 µg/ml BSA 50% Glycerol pH 7.4 @ 25°C Heat Inactivation 65°C for 20 minutes Methylation Sensitivity dam methylation: Not Sensitive dcm methylation: Blocked by Some Combinations of Overlapping CpG Methylation: Blocked by Some Combinations of Overlapping FAQ Q: Is Acc65I an isoschizomer of KpnI? A: Yes, but it leaves a different cohesive end. KpnI produces a 4-base 3´ extension, whereas Acc65I leaves a 4-base 5´ extension. Q: What factors inhibit Acc65I activity? A: Overlapping dcm methylation. Dcm CC(A/T)GG blocks Acc65I when the sequence is GGTACC(A/T)GG or CC(A/T)GGTACC. 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: Yes. This enzyme cannot cut recognition sites that are blocked by some combinations of overlapping dcm methylation or by some combination 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.