New England Biolabs, R0596S, BciVI

BciVI has been reformulated with Recombinant Albumin (rAlbumin) beginning with Lot #10317355. Related Categories Restriction Endonucleases B,, Time-Saver Qualified Restriction Enzymes 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 rCutSmart™ Buffer Incubate at 37°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: 100% NEBuffer™ r2.1: 25% NEBuffer™ r3.1: <10% rCutSmart™ Buffer: 100% Diluent Compatibility Diluent C Storage Buffer 10 mM Tris-HCl 250 mM NaCl 1 mM DTT 0.1 mM EDTA 200 µg/ml Recombinant Albumin 50% Glycerol 0.15% Triton® X-100 pH 7.4 @ 25°C Heat Inactivation 80°C for 20 minutes Methylation Sensitivity dam methylation: Not Sensitive dcm methylation: Not Sensitive CpG Methylation: Not Sensitive FAQ Q: Is BciVI inhibited by salt? A: Activity decreases above 50 mM NaCl. A 70% ethanol wash is recommended for mini-prep DNA. Q: Is extended digestion of BciVI recommended? A: Extended digestion is not beneficial because the enzyme is not stable in reaction. Q: Why is BciVI- cut DNA difficult to ligate? A: Single base-pair overhangs are poor substrates for T4 DNA ligase: they anneal very poorly, and are as difficult to ligate as are blunt ended DNA fragments. Also, for AlwI the overhang is not constrained to be any particular nucleotide; all four bases are roughly equally represented. Consequently, any given overhang is compatible with only 25% of the other overhangs present. This lowers ligation efficieny relative to overhangs generated by restriction enzymes which leave only mutually compatible overhangs. Ligation efficiency can be increased by using our Blunt/TA Ligase Master Mix. Q: Is the recognition site nonpalindromic? A: Yes. The recognition sequence can be read as either GTATCC or GGATAC. 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: 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: 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: 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.