New England Biolabs, R0568S, BfaI

BfaI has been reformulated with Recombinant Albumin (rAlbumin) beginning with Lot #10155313. Related Categories Restriction Endonucleases B 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: <10% NEBuffer™ r2.1: 10% NEBuffer™ r3.1: <10% 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 80°C for 20 minutes Methylation Sensitivity dam methylation: Not Sensitive dcm methylation: Not Sensitive CpG Methylation: Not Sensitive FAQ Q: Does BfaI work well on PCR products? A: No. BfaI has shown minimal cleavage of unpurified PCR products. Therefore, PCR products should be purified prior to BfaI digestion in 1 X rCutSmart Buffer. Q: Does BfaI replace an enzyme previously sold? A: Yes, BfaI replaces RmaI. Q: What is the activity of BfaI at 25°C? A: BfaI is 25-50% active at 25°C. 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: Are the DNA fragments produced by Vent DNA Polymerase blunt-ended or do they have the single-base 3' overhang that Taq DNA Polymerase yields? A: Vent DNA Polymerase generates > 95% blunt-ended fragments. The exo- derivatives are more like Taq DNA Polymerase in that there are predominantly two kinds of ends seen; 70% are blunt-ended, while 30% are predominantly single base 3' 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: 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.