New England Biolabs, R0123S, SfiI

Requires two or more sites for cleavage. Please review the enzyme list and recommendations in the following Related Categories Restriction Endonucleases S,, 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 pXba DNA in 1 hour at 50°C in a total reaction volume of 50 μl. Reaction Conditions 1X rCutSmart™ Buffer Incubate at 50°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: 25% NEBuffer™ r2.1: 100% NEBuffer™ r3.1: 50% rCutSmart™ Buffer: 100% Diluent Compatibility Diluent C Storage Buffer 10 mM Tris-HCl 250 mM NaCl 0.1 mM EDTA 200 µg/ml BSA 50% Glycerol 0.15% Triton® X-100 1 mM DTT pH 7.4 @ 25°C Heat Inactivation No Methylation Sensitivity dam methylation: Not Sensitive dcm methylation: Impaired by Overlapping CpG Methylation: Blocked by Some Combinations of Overlapping Activity at Temperature @37°C: 10% FAQ Q: What is the minimum distance needed between two SfiI sites? Can they follow each other directly? A: This question can be answered at a few levels. If you merely want to achieve an efficiency of digestion as good as in a vector having a single SfiI site (which is an inefficient digestion) then you can use a separation of approximately 10bp. For optimal efficiency of cleavage of the two SfiI sites, you would want to include a ""stuffer fragment"" of at least 170bp. [I would suggest 200bp.] The kinetics of SfiI appear to be unique among commercially available restriction enzymes. For this reason, SfiI reactions can be difficult to optimize. A vector with a single SfiI site is probably the most difficult type of SfiI site if the goal is to achieve absolute completion. Both the ratio of enzyme: DNA (units:micrograms) and the DNA concentration are important. A vector which has two SfiI sites is cut much more rapidly, due to a cooperative reaction involving both sites, BUT ONLY if the the two sites are separated by at least 170bp (enough to allow looping of the DNA.) Q: Can I achieve digestion of a plasmid with a single SfiI site? A: Trans reactions are aided by increasing DNA concentration (so you may achieve digestion by increasing the DNA concentration, but it may not be efficient) but, perversely, SfiI reaction can be hindered by increasing enzyme concentration. In reactions with enzyme in excess of DNA sites, each site becomes loaded with a SfiI tetramer, thus preventing the protein from binding the two sites necessary for full activity. Q: Do degenerate recognition sites need to be palindromic? A: Most restriction enzyme recognition sites are palindromic and include only specified base pairs (i.e., EcoRI recognizes GAATTC). However, some enzymes have degenerate sites, meaning that they contain one or more base pairs that are not specifically defined (i.e., BsrFI-v2 recognizes RCCGGY, where R= A or G and Y= C or T). For degenerate enzymes, any base represented by the single letter code may be present at either location in the recognition site for cleavage to occur. For example, BsrFI-v2 recognizes all of the following sequences: ACCGGC, ACCGGT, GCCGGC, GCCGGT. Q: How can this enzyme be inactivated? A: To inactivate enzymes that cannot be heat killed, we recommend a phenol extraction followed by ethanol precipitation or a commercial spin column designed to purify DNA. Q: Why isn't the enzyme cutting completely? A: Substrates with one SfiI site are cut less efficiently than substrates with two sites. ""SfiI exists in solution as a tetramer and it appears to interact with two copies of it's recognition sequence before it can cleave DNA. It's primary reaction is then to cut both strands at both sites in a concerted process. The two sites can be either on the same or different DNA molecules.""- from Lois M. Wentzell, Timothy J. Nobbs and Stephen E. Halford, Mol. Biol. 248:581-595 (1995). ""The SfiI restriction endonuclease makes a four strand DNA break at two copies of it's recognition sequence. Q: Can I add an oligo to my reaction so that two sites are available for efficient cutting? A: Yes. As long as their are two sites available, whether they are on the same or different DNA molecules does not matter. 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: Is this enzyme sensitive to dam, dcm or mammalian CpG methylation? A: Yes. This enzyme cannot cut recognition sites that are impaired by overlapping dcm methylation or blocked 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.