New England Biolabs, R0703S, BtgZI

Learn about Ligase Fidelity and Push the Limits of Related Categories Restriction Endonucleases B Applications High-throughput cloning and automation solutions,, 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 60°C in a total reaction volume of 50 μl. Reaction Conditions 1X rCutSmart™ Buffer Incubate at 60°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: 10% NEBuffer™ r2.1: 25% NEBuffer™ r3.1: <10% rCutSmart™ Buffer: 100% Diluent Compatibility Diluent A Storage Buffer 10 mM Tris-HCl 50 mM KCl 1 mM DTT 0.1 mM EDTA 200 µg/ml BSA 50% Glycerol pH 7.4 @ 25°C Heat Inactivation 80°C for 20 minutes Methylation Sensitivity dam methylation: Not Sensitive dcm methylation: Not Sensitive CpG Methylation: Impaired Activity at Temperature @37°C: 50% FAQ Q: What is the activity of BtgZI at 37°C? A: BtgZI has 75% activity at 37°C. Q: Is BtgZi activity blocked by methylation? A: Cleavage of mammalian genomic DNA is blocked by CpG methylation. For up-to-date information about methylation sensitivities, please visit Dam-Dcm and CpG Methylation or REBASE. Q: Why are the DNA bands run on an agarose gel smeared? A: BtgZI can remain bound to DNA after cutting and alter the migration rate of DNA during electrophoresis. To disrupt binding, add SDS to a final concentration of 0.5% or purify DNA before electrophoresis. 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: Which restriction enzymes are used in GoldenBraid Assembly? A: BsaI is one Type IIS enzyme used in this method. NEB also offers an engineered high-fidelity version of this enzyme, BsaI-HF®v2, which has the added benefit of being Time-Saver™ qualified (can digest DNA in 5-15 mins and can be digested safely overnight) and exhibits reduced star activity. BsaI-HFv2 works in CutSmart® Buffer, as does T4 DNA Ligase (also part of the GoldenBraid workflow) which is 100% functionally active in this buffer, when the reaction is supplemented with 1 mM ATP. Other Type IIS enzymes used in GoldenBraid include BsmBI-v2/Esp3I, BtgZI, BbsI/BbsI-HF and PaqCI (AarI isoschizomer with 7 bp recognition sequence). Reference: GoldenBraid 2.0: A Comprehensive DNA Assembly Framework for Plant Synthetic Biology Q: Which restriction enzymes are used in Golden Gate Assembly? A: Golden Gate Assembly is a one-tube efficient cloning method based on Type IIS restriction enzymes that cleave outside their recognition sites and leave 3 or 4-base overhangs. BsaI is the most commonly used Type IIS enzyme for Golden Gate Assembly. NEB also offers an engineered high-fidelity version of this enzyme, BsaI-HF®v2, which has the added benefit of being Time-Saver™ qualified (can digest DNA in 5-15 mins or overnight without degradation to DNA) and exhibits dramatically reduced star activity. Other Type IIS enzymes used in Golden Gate Assembly include BsmBI-v2/Esp3I, BbsI/BbsI-HF, PaqCI (AarI isoschizomer with 7 bp recognition sequence), and SapI/BspQI/BspQI-HF (with 7bp recognition sequence and 3bp overhangs). 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.