New England Biolabs, R0197S, ClaI

ClaI has been reformulated with Recombinant Albumin (rAlbumin) beginning with Lot #10119493. Related Categories Restriction Endonucleases C G,, 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 (dam - ) 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: 50% NEBuffer™ r3.1: 50% rCutSmart™ Buffer: 100% Diluent Compatibility Diluent A Storage Buffer 10 mM Tris-HCl 50 mM KCl 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: Blocked by Overlapping dcm methylation: Not Sensitive CpG Methylation: Blocked FAQ Q: Is ClaI sensitive to methylation? A: Yes, it is blocked by overlapping dam methylation, and by CpG methylation at all sites. For up-to-date information about methylation sensitivities, please visit Dam-Dcm and CpG Methylation or REBASE. Q: Does ClaI produce commonly used compatible ends? A: BspDI cleaves to leave a 5´GC which can be ligated to fragments generated by AccI, BspDI, BstBI, TaqI, AciI, BsaHI, HinP1I, HpaII, NarI, Psp1406I. Q: What is the activity of ClaI at 25°C? A: ClaI has 25-50% activity at 25°C. 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: 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.