New England Biolabs, M0663S, T5 Exonuclease

This product replaces T5 Exonuclease (NEB #M0363). Related Categories Exonucleases and Non-specific Endonucleases Applications Gibson Assembly Specification Unit Definition 1 unit of T5 Exonuclease is defined as the amount of enzyme required to cause the change of 0.00032 A260 nm/min at 37° C in rCutSmart Buffer. Reaction Conditions 1X NEBuffer™ 4 Incubate at 37°C 1X NEBuffer™ 4 50 mM Potassium Acetate 20 mM Tris-acetate 10 mM Magnesium Acetate 1 mM DTT (pH 7.9 @ 25°C) Storage Buffer 50 mM Tris-HCl 100 mM NaCl 1 mM DTT 0.1 mM EDTA 50% Glycerol 0.1% Triton® X-100 pH 7.5 @ 25°C Heat Inactivation No Unit Assay Conditions 1X CutSmart Buffer; 40 µg/ml Salmon Sperm DNA FAQ Q: How Does T5 Exonuclease differ from Lambda Exonuclease (NEB# M0262)? A: Unlike Lambda Exonuclease, T5 Exonuclease can degrade nicked-circular dsDNA. T5 Exonuclease also acts on ssDNA, while Lambda Exonuclease does not. Q: Will T5 exonuclease cleave supercoiled dsDNA? A: No. dsDNA exonucleases generally do not cleave supercoiled or closed circular DNA. Exonucleases need an end or a nick to initiate activity. Q: How does T5 Exonuclease differ from Exonuclease III (NEB# M0206)? A: Exonuclease III acts in the 3' to 5' direction, opposite that of T5 Exonuclease, which acts in the 5’ to 3’ direction. Q: What is the activity of T5 Exonuclease in the NEBuffers? A: NEBuffer 1: less than 20% NEBuffer 2: 75% NEBuffer 3: 50% NEBuffer 4: 100% Q: Can T5 Exonuclease be heat inactivated? A: No. Q: Can DNA be blunted using T5 Exonuclease? A: No. Use DNA Polymerase I, Large (Klenow) Fragment (NEB# M0210) to fill in 5' overhangs (also called 3' recessed ends) and chew back 3' overhangs or use Mung Bean Nuclease (NEB# M0250) to chew back 3' or 5' overhangs. Note: 3' overhangs cannot be filled in. Q: Will T5 Exonuclease work in rCutSmart buffer? A: Yes, T5 Exonuclease will work in rCutSmart buffer. To see its % functional activity in rCutSmart, and that of other DNA Modifying enzymes in the cloning workflow, refer to the Activity of DNA Modifying Enzymes in rCutSmart® Buffer chart. Q: What is the difference between T5 Exonuclease (NEB #M0363) and T5 Exonuclease (NEB #M0663)? A: T5 Exonuclease (NEB #M0663) is a His-tagged version of the discontinued T5 Exonuclease (NEB #M0363). The unit definition has been modified to align with the GMP-grade* version of the product. Product performance, protein concentration and reaction conditions remain the same. * “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.