New England Biolabs, R0172S, SfaNI

The large size of this product was discontinued on 12/31/2020. The small size will continue to be available. Related Categories Restriction Endonucleases S Applications Restriction Enzyme Digestion Specification Unit Definition One unit is defined as the amount of enzyme required to digest 1 µg of ΦX174 RF I DNA in 1 hour at 37°C in a total reaction volume of 50 μl. Reaction Conditions 1X NEBuffer™ r3.1 Incubate at 37°C 1X NEBuffer™ r3.1 100 mM NaCl 50 mM Tris-HCl 10 mM MgCl2 100 µg/ml Recombinant Albumin (pH 7.9 @ 25°C) Activity in NEBuffers NEBuffer™ r1.1: <10% NEBuffer™ r2.1: 75% NEBuffer™ r3.1: 100% rCutSmart™ Buffer: 25% Diluent Compatibility Diluent B Storage Buffer 10 mM Tris-HCl 300 mM NaCl 1 mM DTT 0.1 mM EDTA 500 µg/ml BSA 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: Impaired by Some Combinations of Overlapping FAQ Q: Is extended digestion using SfaNI recommended? A: No, the DNA may get degraded. TypeIIs enzymes such as SfaNI will often yield worse results if more enzyme is added than is necessary or if digestion longer than one hour is attempted. This effect is not due to a contaminating nuclease, but instead to inherent additional activities of the SfaNI enzyme. Because this enzyme is composed of distinct recognition and cleavage moieties, random degradation of the protein during a long digest will occasionally release an active cleavage moiety which is no longer regulated by the recognition moiety. Thus, long digests will result in the creation of nuclease. This property is not the same as Star Activity. Q: Why do the bands smear after SfaNI digestion when running an average agarose gel? A: This is due to binding of the enzyme to the DNA, which will alter the migration rate (and/or cause smearing). This can be remedied by adding an ionic detergent (such as 0.1% SDS) to the sample before loading on the gel. Alternatively, extracting the sample with phenol, or using a `quick spin´ DNA purification column can help. This second cause relates to the tendency of some restriction enzymes to remain bound to their substrate after cleavage. All restriction enzymes have a very tight binding affinity for their recognition sites. And, they all have secondary, and relatively lower affinities for DNA in general, and for the half-site which results from cleavage. The strength of this secondary affinity varies greatly among restriction enzymes. For most restriction enzymes, this secondary affinity is sufficiently low enough to not be observed when digestion products are run on a gel. But, for several restriction enzymes, the secondary binding will cause an alteration of migration rate of the DNA fragments. Q: Does SfaNI cleave ssDNA? A: No. 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 in mammalian gDNA that are impaired by some combinations of 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.