New England Biolabs, R3733S, BsaI-HF®v2

GMP-grade reagent also available. Related Categories High-Fidelity (HF®) Restriction Endonucleases,, Restriction Endonucleases B,, Time-Saver Qualified Restriction Enzymes Applications NEBridge® Golden Gate Assembly ,, Restriction Enzyme Digestion,, Fast Cloning: Accelerate your cloning workflows with reagents from NEB, Specification Unit Definition One unit is defined as the amount of enzyme required to digest 1 µg of pXba 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: 100% NEBuffer™ r2.1: 100% NEBuffer™ r3.1: 100% rCutSmart™ Buffer: 100% Diluent Compatibility Diluent B Storage Buffer 20 mM Tris-HCl 300 mM NaCl 0.1 mM TCEP 200 µg/ml Recombinant Albumin 50% Glycerol pH 9 @ 25°C Heat Inactivation 80°C for 20 minutes Methylation Sensitivity dam methylation: Not Sensitive dcm methylation: Impaired by Some Combinations of Overlapping CpG Methylation: Blocked by Some Combinations of Overlapping FAQ 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: When should I choose the HF version of an 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 is more convenient for double digestion or other multi-step protocol. There is no disadvantage to using the HF version. 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: 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 restriction enzymes are used in Golden Gate Assembly? A: Golden Gate Assembly is a one-tube efficient cloning method based on Type IIS restriction enzymes that cleave outside their recognition sites and leave 3 or 4-base overhangs. BsaI is the most commonly used Type IIS enzyme for Golden Gate Assembly. NEB also offers an engineered high-fidelity version of this enzyme, BsaI-HF®v2, which has the added benefit of being Time-Saver™ qualified (can digest DNA in 5-15 mins or overnight without degradation to DNA) and exhibits dramatically reduced star activity. Other Type IIS enzymes used in Golden Gate Assembly include BsmBI-v2/Esp3I, BbsI/BbsI-HF, PaqCI (AarI isoschizomer with 7 bp recognition sequence), and SapI/BspQI/BspQI-HF (with 7bp recognition sequence and 3bp overhangs). Q: Which restriction enzymes are used in GoldenBraid Assembly? A: BsaI is one Type IIS enzyme used in this method. NEB also offers an engineered high-fidelity version of this enzyme, BsaI-HF®v2, which has the added benefit of being Time-Saver™ qualified (can digest DNA in 5-15 mins and can be digested safely overnight) and exhibits reduced star activity. BsaI-HFv2 works in CutSmart® Buffer, as does T4 DNA Ligase (also part of the GoldenBraid workflow) which is 100% functionally active in this buffer, when the reaction is supplemented with 1 mM ATP. Other Type IIS enzymes used in GoldenBraid include BsmBI-v2/Esp3I, BtgZI, BbsI/BbsI-HF and PaqCI (AarI isoschizomer with 7 bp recognition sequence). Reference: GoldenBraid 2.0: A Comprehensive DNA Assembly Framework for Plant Synthetic Biology 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: How many nucleotides do I have to add adjacent to the RE recognition site in order to get efficient cutting? A: Restriction Enzyme Digest Protocol: Cutting Close to DNA End 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: What is the activity of the Type IIS restriction enzyme BsaI-HFv2 (NEB #R3733) in T4 DNA Ligase Buffer? A: BsaI-HFv2 exhibits 100% activity in T4 DNA Ligase Buffer. In contrast, BsaI (NEB #R0535) has only 50% activity in T4 DNA Ligase Buffer. Q: Is this enzyme sensitive to dam, dcm or mammalian CpG methylation? A: Yes. This enzyme cannot cut recognition sites that are impaired by some combinations of 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: Does NEB offer any BSA-free and/or animal origin-free restriction enzymes for linearization of plasmids for mRNA vaccine development? A: Yes, NEB has formulated some restriction enzymes (including BspQI, an SapI isoschizomer) without animal-derived components and no BSA appears in their final formulation. In addition, we have several restriction enzymes available without BSA nor other animal-derived components in their final formulation. This list includes: BbsI-HF BsaI-HFv2 BspQI, isoschizomer of LguI, GMP-grade* now available BspQI-HF ClaI HindIII-HF PacI SapI SpeI SwaI XbaI XhoI XmnI Other enzymes can be formulated upon request. Please inquire here. * “GMP Grade” and “GMP-grade” are branding terms NEB uses to describe products manufactured or finished at NEB’s Rowley facility. The Rowley facility was designed to manufacture products under more rigorous infrastructure and process controls to achieve more stringent product specifications and customer requirements. Products manufactured at NEB’s Rowley facility are manufactured in compliance with ISO 9001 and ISO 13485 quality management system standards. However, at this time, NEB does not manufacture or sell products known as Active Pharmaceutical Ingredients (APIs), nor does NEB manufacture its products in compliance with all of the Current Good Manufacturing Practice regulations. 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: 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. 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