New England Biolabs, R0149T, TaqI-v2

Related Categories Restriction Endonucleases T Z,, 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 65°C in a total reaction volume of 50 μl. Reaction Conditions 1X rCutSmart™ Buffer Incubate at 65°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: 50% NEBuffer™ r2.1: 100% NEBuffer™ r3.1: 50% rCutSmart™ Buffer: 100% Diluent Compatibility Diluent B Storage Buffer 10 mM Tris-HCl 300 mM NaCl 1 mM DTT 0.1 mM EDTA 500 µg/ml Recombinant Albumin 50% Glycerol pH 7.4 @ 25°C Heat Inactivation No Methylation Sensitivity dam methylation: Blocked by Overlapping dcm methylation: Not Sensitive CpG Methylation: Not Sensitive Activity at Temperature @37°C: 10% FAQ Q: Why isn't TaqI-v2 cutting? A: 1. It could be inhibited by some mini-prep impurities. Drop-dialyse or ethanol wash. 2. It is blocked by overlapping dam methylation. 3. PCR products can be resistant to cleavage. Purify the DNA. 4. Incubation without BSA results in 50% activity. 5. Incubation at 37°C results in 10% activity. Q: Does a certain pH range inhibit TaqI-v2  activity? A: Yes, activity is decreased if the reaction buffer pH is not between 7.5 and 8.5. Q: Does TaqI-v2 recognize mutliple sites? A: The enzyme can recognise A/C or G/T mismatches (NAR 14: 459-6590: 1943-1949 (1986) Jiricny, J. Martin, D. ). The star sites are a single base pair difference (Gene 65: 149-165, (1988) Barany, F.). Q: Is this enzyme sensitive to dam, dcm or mammalian CpG methylation? A: Yes. This enzyme cannot cut recognition sites that are blocked by overlapping dam 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.