New England Biolabs, R3189M, NotI-HF®

NotI-HF has been reformulated with Recombinant Albumin (rAlbumin) beginning with Lot #10133990. Related Categories Restriction Endonucleases: N-O,, High-Fidelity (HF®) Restriction Endonucleases,, 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 pBC4 DNA 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: 25% NEBuffer™ r2.1: 100% NEBuffer™ r3.1: 25% rCutSmart™ Buffer: 100% Diluent Compatibility Diluent A Storage Buffer 10 mM Tris-HCl 50 mM NaCl 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: Not Sensitive dcm methylation: Not Sensitive CpG Methylation: Blocked FAQ Q: Is there any difference in the methylation sensitivity between NotI-HF and NotI? A: Both NotI-HF and NotI perform the same when tested on various CpG methylation patterns. For more specific information regarding methylation, follow the link to the Restriction Enzyme database REBASE. Q: Is there a difference in cutting close to the ends between NotI-HF and NotI? A: No. When tested on a series of five oligos (19-23 bases in length) containing the restriction site and an additional 1 to 5 A/T bases from the end, both enzymes cut the oligo with 1 base pair from the end. When designing primers, NEB recommends adding 6 extra bases to ensure against experimental failure due to variation in substrate and reaction conditions. Q: What is the difference between NotI-HF and NotI? A: NotI-HF has been engineered with a mutation to exhibit significantly less star activity and comes supplied with rCutSmart Buffer. Q: What does HF® refer to following the name of a restriction enzyme? A: HF® stands for high fidelity. Many restriction endonucleases are capable of cleaving sequences which are similar but not identical to their defined recognition sequence. This altered or relaxed specificity has been termed star activity. HF restriction endonucleases have been engineered to cleave with higher fidelity than the wild type enzyme, hence exhibiting less star activity. Screens using increased glycerol concentration, increased reaction time and high enzyme concentration were used to identify enzymes that would offer the highest fidelity over a wide range of conditions In addition, all HF enzymes are supplied with rCutSmart Buffer™ as well as a free vial of Purple Loading Dye. Learn more about High Fidelity Restriction Enzymes here. Q: When is star activity a concern? A: Star activity is of concern if extra banding can cause misinterpretation of results in genotyping and mutational analysis procedures. Experimental design can promote star activity. Small reaction volumes are more likely to contain glycerol concentrations of 5% or greater, a condition known to increase star activity. A 5% glycerol concentration occurs when setting up a double digest in a 20 µl reaction using 1 µl of each enzyme. Overnight digests are more likely to generate star activity. For tips on avoiding star activity, please click here. Q: Why does the HF version of the enzyme have a different recommended buffer than the wild type enzyme? A: In many cases, changing the charged amino acids of a restriction endonuclease gene results in changes in buffer preference. These changes can be significant. For example, wild type SalI has a strict requirement for NEBuffer r3.1, a high ionic strength buffer. However, SalI-HF works well in NEBuffer r2.1 and rCutSmart Buffer™, which are moderate ionic strength buffers. Q: When should I choose the High Fidelity (HF®) version of the enzyme? A: The HF version of the enzyme has the same cleavage specificity as the wild type enzyme, and should be chosen if star activity is a concern or if the recommended buffer, CutSmart®, is more convenient for double digestion or other another multi-step protocol. In some instances, HF enzymes may have different heat inactivation temperature (or time), salt tolerance and (rarely) methylation properties. Learn more about High Fidelity Restriction Enzymes. Q: Can the change in buffer preference of the HF enzyme be advantageous? A: All HF restriction enzymes are supplied with rCutSmart® Buffer. The HF enzymes typically have a different optimal buffer than the wild type enzyme; this can be advantageous when designing double digest experiments. rCutSmart Buffer was chosen as the recommended buffer for HF enzymes since it shows the highest level of enzyme compatibility in the NEB buffer system. Q: Will the HF enzyme produce elevated star activity when used in a buffer other than the one recommended? A: Star activity was extensively tested in all NEBuffers that showed at least 50% enzyme activity. Star activity is significantly reduced compared to the wild type enzyme activity in all NEBuffers. However, we recommend using the suggested reaction buffer when possible because there are cases where star activity can still be problematic under extreme conditions using non-recommended buffers. Q: What does it mean to be Time-Saver™ qualified? A: Enzymes that are Time-Saver qualified will digest unit assay substrate in 5-15 minutes under recommended reaction conditions, and can also be used safely in overnight digestions. Q: How is the improvement in fidelity of HF restriction endonucleases quantitated? A: Star activity is measured using a Fidelity Index (FI). The FI of the wild type restriction endonuclease can be directly compared to the modified HF version. Q: What is the Fidelity Index (FI)? A: “The Fidelity Index (FI) is defined as the ratio of the maximum enzyme amount showing no star activity to the minimum amount needed for complete digestion at the cognate recognition site for any particular restriction endonuclease.” The FI value translates into the minimum number of units that was required to produce star activity. The FI value can be influenced by substrate, buffer and additives like glycerol.The following reference explains how the Fidelity Index is determined. Hua Wei et. al. “The Fidelity Index provides a systematic quantitation of star activity of DNA restriction endonucleases”, Nucleic Acids Research, 2008, Vol.36, No. 9: e50. 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: 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. 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 blocked by 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: Is Gel Loading Dye, Purple (6X) or Gel Loading Dye, Purple (6X), no SDS compatible with other DNA binding dyes such as SYBR® and GelRed™ during gel electrophoresis? A: Because high affinity nucleic acid binding dyes can affect DNA migration during electrophoresis, post-staining of gels with SYBR or GelRed dyes is highly recommended. However, these dyes can also be used as precast dyes (into the agarose gel). Because the Gel Loading Dye, Purple (6X) has an increased concentration in SDS, some interference may be observed when using SYBR or GelRed as precast dyes. When using these dyes as precast dyes, NEB recommends using our Gel Loading Dye, Purple, No SDS (6X) (NEB #B7025S ) instead. Please follow the recommendations below for both Purple dyes when using SYBR dyes as precast dyes: Reduce the amount of sample DNA loaded to 250 to 500ng, and use 0.5X only of SYBR dyes in precast gels. These dyes are much more sensitive than EtBr. Blown out or smeared bands can be caused by overloading. If using Gel Loading Dye, Purple (6X) B7024S , use TBE instead of TAE buffer, as the SDS interference is increased when using TAE buffer (in the gel and as a running buffer). Use the SYBR dyes as recommended by the manufacturer: add the SYBR dye to the TBE buffer first, then add the SYBR dye/TBE mix to the molten agarose solution. It is preferable to wait a few minutes to add the SYBR dyes to the molten agarose. Adding it to a cooled agarose solution (above gelling temperature, 45°C) produces better results. Always run a freshly made agarose gel, for a single run only. Re-running the gel will yield poor result. Please follow the recommendations below for both Purple dyes when using GelRed dyes as precast dyes: Reduce the amount of sample DNA loaded to 60 to 125ng, and use 0.5X only of GelRed dye in precast gels. This dye is much more sensitive than EtBr. Blown out or smeared bands can be caused by overloading. It is preferable to wait a few minutes to add the GelRed dye to the molten agarose. Adding it to a cooled agarose solution (above gelling temperature, 45°C) produces better results. Always run a freshly made agarose gel, for a single run only. Re-running the gel will yield poor result. SYBR® is a registered trademark of Life Technologies Corporation GelRed™ is a trademark of Biotium