New England Biolabs, R0147L, MboI

MboI has been reformulated with Recombinant Albumin (rAlbumin) beginning with Lot #10149199. Related Categories Restriction Endonucleases H M,, 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 (dam-) 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: 75% NEBuffer™ r2.1: 100% NEBuffer™ r3.1: 100% 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 Recombinant Albumin 50% Glycerol pH 7.4 @ 25°C Heat Inactivation 65°C for 20 minutes Methylation Sensitivity dam methylation: Blocked dcm methylation: Not Sensitive CpG Methylation: Not Sensitive FAQ Q: What's the difference between DpnI, DpnII, MboI, and Sau3AI? A: They all recognize the same sequence but have different methylation sensitivities. DpnI will only cleave fully-adenomethylated dam sites and hemi-adenomethylated dam sites 60X more slowly. DpnII and MboI share methylation sensitivity and cleave dam sites which lack adenomethylation; each is blocked by complete dam methylation and probably by hemi-methylation as well. Sau3AI will cleave all dam sites regardless of adenomethylation but is completely blocked by methylated cytosines within the dam sequence. 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: 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: Is this enzyme sensitive to dam, dcm or mammalian CpG methylation? A: Yes. This enzyme cannot cut recognition sites that are blocked by dam methylation or impaired by overlapping 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: Can Gel Loading Dye, Purple 6X (B7024) be stored in cold temperatures? A: The recommended storage temperature for Gel Loading Dye, Purple 6X, is room temperature (25°C). Commonly, the SDS may precipitate out of solution when the dye is stored on cold temperatures. This slight precipitate can be dissipated by letting the vial sit at room temperature for an hour, or warming gently for 10 minutes. Mix the vial a few times before use, to bring the solution back to homogeneity.