New England Biolabs, R0141L, SmaI

SmaI has been reformulated with Recombinant Albumin (rAlbumin) beginning with Lot #10156748. Related Categories Restriction Endonucleases S,, Time-Saver Qualified Restriction Enzymes Applications 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 (HindIII digest) 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 ml 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: Blocked Activity at Temperature @25°C: 100% FAQ Q: Why isn't SmaI cutting? A: 1. SmaI needs K+ to work. Use in rCutSmart Buffer, or supplement with KCl. 2. SmaI is blocked by CpG methylation. 3. For extended digestions (>1 hour), incubate at 25°C. Q: Is SmaI affected by methylation? A: SmaI is blocked by CpG methylation. For up-to-date information about methylation sensitivities, please visit Dam-Dcm and CpG Methylation or REBASE. Q: How does SmaI differ from its isoschizomer, XmaI? A: XmaI leaves a 5´ extension wheras SmaI leaves blunt ends. XmaI is not blocked by CpG methylation. Q: What is the molecular weight of SmaI? A: The molecular weight of SmaI is 28,634 based on sequence data. 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: 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: 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: In an effort to standardize reaction conditions, the recommended incubation temperature has changed for ApaI (NEB #R0114), BaeI (NEB #R0613), BsrDI (NEB #R0574) and SmaI (NEB #R0141). Which incubation temperature should I use? A: You can safely incubate at either temperature. Please note that for SmaI, we suggest incubating at 25°C if you are doing extended digestions (>1 hour). Q: Are certain restriction enzymes more active at higher incubation temperatures than what is recommended? A: NEB restriction enzymes are 100% active at the recommended incubation temperature in the recommended buffer provided with each enzyme. With some restriction enzymes isolated from thermophilic species, you may see increased activity at higher incubation temperatures. We do not recommend increasing the temperature as additional nuclease activities may appear at non-recommended temperatures.