New England Biolabs, R0539L, BbsI

Learn about Ligase Fidelity and Push the Limits of Related Categories Restriction Endonucleases B,, Time-Saver Qualified Restriction Enzymes Applications NEBridge® Golden Gate Assembly ,, 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 37°C in a total reaction volume of 50 μl. Reaction Conditions 1X NEBuffer™ r2.1 Incubate at 37°C 1X NEBuffer™ r2.1 50 mM NaCl 10 mM Tris-HCl 10 mM MgCl2 100 µg/ml Recombinant Albumin (pH 7.9 @ 25°C) Activity in NEBuffers NEBuffer™ r1.1: 100% NEBuffer™ r2.1: 100% NEBuffer™ r3.1: 25% rCutSmart™ Buffer: 75% 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 65°C for 20 minutes Methylation Sensitivity dam methylation: Not Sensitive dcm methylation: Not Sensitive CpG Methylation: Not Sensitive FAQ 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: What is the activity of BbsI at 25°C? A: BbsI is approximately 25% active at 25°C. Q: Which NEB restriction enzymes are supplied with Gel Loading Dye, Purple (6X)? A: All HF-restriction enzymes and most NEB non-HF restriction enzymes are supplied with Gel Loading Dye, Purple (6X). The non-HF restriction enzymes that come supplied with purple dye are: AatII AsiSI BsmBI-v2 EagI MboI NlaIII PvuI SmaI Acil AvrII BspHI EcoRI MboII NotI RsaI SpeI AfeI BamHI BsrGI EcoRV MluI NspI SacI StuI AflII BbsI ClaI Esp3I FseI MseI PacI SacII AgeI BglII DdeI HaeIII MspI PciI SalI XbaI AluI BsaI DpnI HindIII NcoI PmeI SapI XhoI ApaI BseYI DpnII HpaI NdeI PsiI-v2 SfaNI XmaI ApeKI BsiWI DraI KpnI NheI PstI SfiI XmnI AscI * All HF restriction enzymes are also supplied with Gel Loading Dye, Purple (6X) 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: 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. Q: What are the storage conditions for BbsI? A: BbsI can be stored at either -20°C or -80°C. Q: For enzymes stored at -80°C, is enzyme activity affected after multiple freeze / thaw cycles? A: The following enzymes maintained full activity when tested for 10 freeze-thaw cycles. If you require more than 10 freeze-thaw cycles, we recommend aliquoting the enzyme into smaller volumes and freezing each aliquot at -80C. Cac8I EagI-HF I-SceI KasI NlaIII Please note: FseI maintained full activity for only 5 freeze/thaw cycles.