New England Biolabs, R0154S, HgaI

HgaI has been reformulated with Recombinant Albumin (rAlbumin) beginning with Lot #10274062. Related Categories Restriction Endonucleases H M Applications Restriction Enzyme Digestion Specification Unit Definition One unit is defined as the amount of enzyme required to digest 1 µg of ΦX174 DNA in 1 hour at 37°C in a total reaction volume of 50 μl. Reaction Conditions 1X NEBuffer™ r1.1 Incubate at 37°C 1X NEBuffer™ r1.1 10 mM Bis-Tris-Propane-HCl 10 mM MgCl2 100 µg/ml Recombinant Albumin (pH 7 @ 25°C) Activity in NEBuffers NEBuffer™ r1.1: 100% NEBuffer™ r2.1: 100% NEBuffer™ r3.1: 25% rCutSmart™ Buffer: 100% Diluent Compatibility Diluent A Storage Buffer 10 mM Tris-HCl 50 mM KCl 1 mM DTT 0.1 mM EDTA 50% Glycerol 200 µg/ml Recombinant Albumin pH 7.4 @ 25°C Heat Inactivation 65°C for 20 minutes Methylation Sensitivity dam methylation: Not Sensitive dcm methylation: Not Sensitive CpG Methylation: Blocked FAQ Q: Is HgaI activity sensitive to dam, dcm or mammalian CpG methylation? A: Yes. HgaI may not cut recognition sites in mammalian gDNA that contain CpG methylation. For up-to-date information about methylation sensitivities, please visit Dam-Dcm and CpG Methylation or REBASE. Q: How many base pairs should be added at the end of a PCR primer after the HgaI recognition site to guarantee that HgaI will cut properly? A: The addition of 6 base pairs 5’ of the recognition site is recommended. Q: Does HgaI have any neoschizomers? A: No. For the most current information about isoschizomers/neoschizomers please refer to REBASE. Q: Does HgaI exhibit star activity? A: Under certain reaction conditions such as low ionic strength, high enzyme concentration, glycerol concentration >5% or pH> 8.0HgaI can exhibit star activity. Overdigestions with >5 units of HgaI per µg of DNA and incubations > 1 hour are not recommended. 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.